Apparatus and method for quantitative characterization of a light detector

ABSTRACT

Aspects of the present disclosure include methods for determining a parameter of a photodetector (e.g., a photodetector in a particle analyzer). Methods according to certain embodiments include irradiating a photodetector positioned in a particle analyzer with a light source (e.g., a continuous wave light source) at a first intensity for a first predetermined time interval, irradiating the photodetector with the light source at a second intensity for a second predetermined time interval, integrating data signals from the photodetector over a period of time that includes the first predetermined interval and the second predetermined interval and determining one or more parameters of the photodetector based on the integrated data signals. Systems (e.g., particle analyzers) having light source and a photodetector for practicing the subject methods are also described. Non-transitory computer readable storage medium having instructions stored thereon for determining a parameter of a photodetector according to the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. Provisional Patent Application Ser.No. 63/012,765 filed Apr. 20, 2020; the disclosure of which applicationis herein incorporated by reference.

INTRODUCTION

The characterization of analytes in biological fluids has become animportant part of medical diagnoses and assessments of overall healthand wellness of a patient. Detecting analytes in biological fluids, suchas human blood or blood derived products, can provide results that mayplay a role in determining a treatment protocol of a patient having avariety of disease conditions.

Flow cytometry is a technique used to characterize and often times sortbiological material, such as cells of a blood sample or particles ofinterest in another type of biological or chemical sample. A flowcytometer typically includes a sample reservoir for receiving a fluidsample, such as a blood sample, and a sheath reservoir containing asheath fluid. The flow cytometer transports the particles (includingcells) in the fluid sample as a cell stream to a flow cell, while alsodirecting the sheath fluid to the flow cell. To characterize thecomponents of the flow stream, the flow stream is irradiated with light.Variations in the materials in the flow stream, such as morphologies orthe presence of fluorescent labels, may cause variations in the observedlight and these variations allow for characterization and separation.

To characterize the components in the flow stream, light must impinge onthe flow stream and be collected. Light sources in flow cytometers canvary from broad spectrum lamps, light emitting diodes as well as singlewavelength lasers. The light source is aligned with the flow stream andan optical response from the illuminated particles is collected andquantified.

SUMMARY

Aspects of the present disclosure include methods for determining aparameter of a photodetector (e.g., a photodetector in a particleanalyzer). Methods according to certain embodiments include irradiatinga photodetector with a light source at a first intensity for a firstpredetermined time interval, irradiating the photodetector with thelight source at a second intensity for a second predetermined timeinterval, integrating data signals from the photodetector over a periodof time that includes the first predetermined interval and the secondpredetermined interval and determining one or more parameters of thephotodetector based on the integrated data signals. In some instances,the light source is a continuous wave light source. In some instances,the light source is a pulsed light source. In some embodiments, thelight source is a light emitting diode. In certain instances, the lightsource is a narrow bandwidth light source, such as a light source whichemits light having wavelengths that span 20 nm or less.

In practicing the subject methods according to certain embodiments, theintensity of the light source is increased in intensity after eachpredetermined time interval (i.e., the second irradiation intensity isgreater than the first irradiation intensity). The time interval forirradiating the photodetector at each light intensity may vary. In someinstances, each time interval is the same. In other instances, each timeinterval is different. In certain embodiments, methods includeirradiating the photodetector with the continuous wave light source at aplurality of intensities over a plurality of predetermined timeintervals. In these embodiments, some of the time intervals may be thesame and some of the time intervals may be different. In some instances,methods include increasing the intensity of light from the light sourceover a period of time that includes at least the first predeterminedinterval and the second predetermined interval. In some instances, theintensity of light from the light source is linearly increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval. In other instances, the intensityof light from the light source is exponentially increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval.

In embodiments, methods include integrating data signals from thephotodetector over a period of time that includes at least the timeintervals of irradiation at each different light intensity. In someembodiments, data signals from the photodetector are integrated over atime period that includes a first time interval where the light sourceirradiates the photodetector at a first intensity and a second timeinterval where the light source irradiates the photodetector at thesecond intensity. In other embodiments, data signals from thephotodetector are integrated over a period of time that includes aplurality of time intervals where the light source irradiates thephotodetector at increasing light intensities during each of theplurality of time intervals.

Integrating data signals from the photodetector in certain embodimentsincludes calculating a signal amplitude over the period of time. In someinstances, calculating the signal amplitude includes calculating one ormore of the median signal amplitude, mean signal amplitude, the standarddeviation in the signal amplitudes and the variance and coefficient ofvariation of the signal amplitude. In certain instances, methods alsoinclude comparing the calculated signal amplitude with the lightintensity of the light source. Based on one or more of the calculatedsignal amplitude and the comparison between the calculated signalamplitude with the light intensity of the light source, a parameter ofthe photodetector is calculated. For instance, methods may includedetermining for the photodetector a parameter such as minimum detectionthreshold, maximal detection threshold, detector sensitivity, detectordynamic range, detector signal-to-noise ratio or number ofphotoelectrons per unit output. The detector parameter may be determinedover a range of operating voltages of the photodetector, such as wherethe parameters of the photodetector are determined over the entireoperating voltage range of the photodetector. In certain embodiments,the photodetector is positioned in a particle analyzer, such as wherethe photodetector is a part of a light detection module of a particleanalyzer. In some instances, the particle analyzer is incorporated intoa flow cytometer where the photodetector is positioned to detect lightfrom particles in a flow stream.

In some embodiments, methods include determining one or more parametersof a photodetector (e.g., a photodetector in a particle analyzer) byirradiating particles in a flow stream where the particles include oneor more fluorophores. In some instances, the particles are beads (e.g.,polystyrene beads). In some instances, methods for determining aparameter of a photodetector include irradiating a flow stream havingparticles that include one or more fluorophores at a first intensity fora first predetermined time interval and at a second intensity for asecond predetermined time interval, detecting light from the flow streamwith the photodetector with a light source, generating a data signalfrom the photodetector at the first irradiation intensity and generatinga data signal from the photodetector at the second irradiation intensityand determining one or more parameters of the photodetector based on thedata signals generated at the first intensity and the second intensity.In some instances, methods include determining the mean fluorescenceintensity from the particles at the first irradiation intensity and atthe second irradiation intensity. In some instances, methods includedetermining the variance of the mean fluorescence intensity at the firstirradiation intensity and at the second irradiation intensity. In someinstances, methods include determining the statistical photo electrons(SPE) at the first irradiation intensity and at the second irradiationintensity. In certain instances, methods further include calculatingdetector efficiency (Q_(det)) of the photodetector for each fluorophoreon the particle based on the statistical photo electrons and thedetermined mean fluorescence intensity of the fluorophore. In certainembodiments, methods include determining the detector efficiency foreach detector channel of the photodetector. In some embodiments, methodsfurther include determining a background signal of each photodetector.In some embodiments, methods further include determining the electronicnoise from each photodetector. In certain embodiments, method furtherinclude determining a detection limit of the photodetector.

Aspects of the present disclosure also include systems having a lightsource and a photodetector that is configured to detect light from thelight source at a first intensity for a first predetermined timeinterval and detect light from the light source at a second intensityfor a second predetermined time interval; and a processor with memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to integrate data signals from the photodetector over aperiod of time that includes the first predetermined time interval andthe second time interval; and determine one or more parameters of thephotodetector based on the integrated data signals. In some embodiments,the system is a particle analyzer. In some instances, the photodetectoris a part of a light detection module of the particle analyzer. Incertain instances, the particle analyzer is incorporated into a flowcytometer.

The light source of the subject systems is, in some embodiments, acontinuous wave light source. In other embodiments, the light source isa pulsed light source. In certain embodiments, the light source is alight emitting diode. In some instances, the light source is a narrowbandwidth light source, such as a light emitting diode that emits lighthaving wavelengths that span 20 nm or less. The light source isconfigured to irradiate a photodetector at two or more differentintensities for predetermined time intervals. In some embodiments, thelight source is configured to irradiate the photodetector at a firstintensity for a first predetermined time interval and to irradiate thephotodetector at a second intensity for a second predetermined timeinterval. In other embodiments, the light source is configured toirradiate the photodetector a plurality of intensities over a pluralityof predetermined time intervals. In embodiments, each time interval maybe the same duration or a different duration. In some embodiments, thelight source is configured to increase the intensity of irradiationafter each predetermined time interval (i.e., the intensity of light isincreased for each successive interval of irradiation). In someembodiments, the light source is configured to increase in intensityover a period of time that includes at least the first predeterminedtime interval and the second predetermined time interval. In someinstances, the intensity of the light source is configured to increaselinearly. In other instances, the intensity of the light source isconfigured to increase exponentially.

Systems of interest include a processor having memory operably coupledto the processor where the memory includes instructions stored thereon,which when executed by the processor, cause the processor to integratedata signals from the photodetector. In some embodiments, the memoryincludes instructions for calculating a signal amplitude from thephotodetector over each time interval. In other embodiments, the memoryincludes instructions for calculating a median signal amplitude. Incertain embodiments, the memory includes instructions for comparing thecalculated signal amplitude with the intensity of irradiation duringeach predetermined time interval.

In embodiments, systems include memory operably coupled to the processorwhere the memory includes instructions stored thereon, which whenexecuted by the processor, cause the processor to determine a parameterof the photodetector based on one or more of the calculated signalamplitude and the comparison between the calculated signal amplitudewith the light intensity of the light source. For instance, the memorymay include instructions for determining minimum detection threshold,maximal detection threshold, detector sensitivity, detector dynamicrange, detector signal-to-noise ratio or number of photoelectrons perunit output. The subject systems may be configured to determine thedetector parameter over a range of operating voltages of thephotodetector, such as over the entire operating voltage range of thephotodetector.

Aspects of the present disclosure also include non-transitory computerreadable storage medium for determining one or more parameters of aphotodetector. In embodiments, the non-transitory computer readablestorage medium includes algorithm for irradiating a photodetector with alight source at a first intensity for a first predetermined timeinterval; algorithm for irradiating the photodetector with the lightsource at a second intensity for a second predetermined time interval;algorithm for integrating data signals from the photodetector over aperiod of time comprising the first predetermined interval and thesecond predetermined interval; and algorithm for determining one or moreparameters of the photodetector based on the integrated data signals. Incertain instances, the non-transitory computer readable storage mediumincludes algorithm for irradiating the photodetector with a plurality oflight intensities over a plurality of time intervals. In theseinstances, the non-transitory computer readable storage medium includesalgorithm for integrating data signals from the photodetector over atime period that includes the plurality of irradiation time intervals.

In some embodiments, the non-transitory computer readable storage mediumincludes algorithm for calculating a signal amplitude. In someinstances, the non-transitory computer readable storage medium includesalgorithm for calculating one or more of the median signal amplitude,mean signal amplitude, the standard deviation in the signal amplitudesand the variance and coefficient of variation of the signal amplitude.In certain instances, the non-transitory computer readable storagemedium includes algorithm for comparing the calculated signal amplitudewith the light intensity of the light source. In certain instance, thenon-transitory computer readable storage medium includes algorithm fordetermining a parameter of the photodetector based on one or more of thecalculated signal amplitude and the comparison between the calculatedsignal amplitude with the light intensity of the light source. Forexample, the non-transitory computer readable storage medium may includealgorithm for determining minimum detection threshold, maximal detectionthreshold, detector sensitivity, detector dynamic range, detectorsignal-to-noise ratio or number of photoelectrons per unit output. Thenon-transitory computer readable storage medium may include algorithmfor determining the detector parameter over a range of operatingvoltages of the photodetector, such as where the parameters of thephotodetector are determined over the entire operating voltage range ofthe photodetector.

In certain embodiments, aspects of the present disclosure also includemultispectral particles (e.g., beads) having one or more fluorophoresfor practicing one or more of the subject methods. Multispectralparticles according to some embodiments include one or morefluorophores, such as 2 or more, such as 3 or more, such as 5 or moreand including 10 or more fluorophores. In some instances, particles ofinterest include a single-peak multi-fluorophore bead that provides fora bright photodetector signal across all light source wavelengths (e.g.,across all LEDs or lasers of the system) and across detectionwavelengths of the photodetectors.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIGS. 1A and 1B depict the measurement of changing light intensity froma light source over a plurality discrete time intervals by aphotodetector according to certain embodiments. FIG. 1A depicts a50-step light intensity ramp spanning 2601 ms. FIG. 1B depicts the first5-steps (spanning 250 ms) of the 50-step light intensity ramp of FIG.1A.

FIG. 2 depicts the measurement of light intensity that changescontinuously according to certain embodiments.

FIG. 3A depicts a flow chart for determining one or more parameters of aphotodetector according to certain embodiments. FIG. 3B depicts a plotused for setting up an initial detector gain for a photodetectoraccording to certain embodiments.

FIG. 4A depicts a functional block diagram of a particle analysis systemfor computational based sample analysis and particle characterizationaccording to certain embodiments. FIG. 4B depicts a flow cytometeraccording to certain embodiments.

FIG. 5 depicts a functional block diagram for one example of a particleanalyzer system according to certain embodiments.

FIG. 6A depicts a schematic drawing of a particle sorter systemaccording to certain embodiments.

FIG. 6B depicts a schematic drawing of a particle sorter systemaccording to certain embodiments.

FIG. 7 depicts a block diagram of a computing system according tocertain embodiments.

DETAILED DESCRIPTION

Aspects of the present disclosure include methods for determining aparameter of a photodetector (e.g., a photodetector in a particleanalyzer). Methods according to certain embodiments include irradiatinga photodetector positioned in a particle analyzer with a light source(e.g., a continuous wave light source) at a first intensity for a firstpredetermined time interval, irradiating the photodetector with thelight source at a second intensity for a second predetermined timeinterval, integrating data signals from the photodetector over a periodof time that includes the first predetermined interval and the secondpredetermined interval and determining one or more parameters of thephotodetector based on the integrated data signals. Systems (e.g.,particle analyzers) having a light source and a photodetector forpracticing the subject methods are also described. Non-transitorycomputer readable storage medium having instructions stored thereon fordetermining a parameter of a photodetector according to the subjectmethods are also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

As summarized above, the present disclosure provides methods and systemsfor determining one or more parameters of a photodetector (e.g., aphotodetector of a particle analyzer). In further describing embodimentsof the disclosure, methods including irradiating a photodetector at afirst intensity over a first predetermined time interval and at a secondintensity over a second predetermined time interval and integrating datasignals from the photodetector over a time period that includes thefirst and second predetermined time intervals are first described ingreater detail. Next, systems (e.g., particle analyzers) having a lightsource and a photodetector for practicing the subject methods aredescribed. Non-transitory computer readable storage medium havinginstructions stored thereon for determining a parameter of aphotodetector according to the subject methods are also provided.

Methods for Determining a Parameter of a Photodetector

Aspects of the present disclosure include methods for determiningparameters of a photodetector (e.g., a photodetector in a particleanalyzer). In practicing the subject methods, a photodetector isirradiated with a light source at a first intensity for a firstpredetermined time interval followed by irradiating the photodetectorwith the light source at a second intensity for a second predeterminedtime interval. In certain embodiments, the light source is a continuouslight source. The term “continuous light source” is used herein in itsconventional sense to refer to a source of light which providesuninterrupted light flux and maintains irradiation of the photodetectorwith little to no undesired changes in light intensity. In someembodiments, the continuous light source emits non-pulsed ornon-stroboscopic irradiation. In certain embodiments, the continuouslight source provides for substantially constant emitted lightintensity. For instance, the continuous light source may provide foremitted light intensity during a time interval of irradiation thatvaries by 10% or less, such as by 9% or less, such as by 8% or less,such as by 7% or less, such as by 6% or less, such as by 5% or less,such as by 4% or less, such as by 3% or less, such as by 2% or less,such as by 1% or less, such as by 0.5% or less, such as by 0.1% or less,such as by 0.01% or less, such as by 0.001% or less, such as by 0.0001%or less, such as by 0.00001% or less and including where the emittedlight intensity during a time interval of irradiation varies by0.000001% or less. The intensity of light output can be measured withany convenient protocol, including but not limited to, a scanning slitprofiler, a charge coupled device (CCD, such as an intensified chargecoupled device, ICCD), a positioning sensor, power sensor (e.g., athermopile power sensor), optical power sensor, energy meter, digitallaser photometer, a laser diode detector, among other types ofphotodetectors.

In certain instances, the light source is a pulsed light source. Theterm “pulsed light source” is used herein in its conventional sense torefer to a source of light which provides light flux in predeterminedintervals, such as by stroboscopic irradiation. The pulse duration mayvary depending on the type of the light source and may be 0.001 ns ormore, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 nsor more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns ormore, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more,such as 10 ns or more, such as 25 ns or more, such as 50 ns or more,such as 100 ns or more, such as 500 ns or more, such as 1000 ns or moreand including a pulse duration of 5 μs or more. For example, the pulseduration of pulsed light sources may range from 0.00001 μs to 1000 μs,such as from 0.00005 μs to 900 μs, such as from 0.0001 μs to 800 μs,such as from 0.0005 μs to 700 μs, such as from 0.001 μs to 600 μs, suchas from 0.005 μs to 500 μs, such as from 0.01 μs to 400 μs, such as from0.05 μs to 300 μs, such as from 0.1 μs to 200 μs and including a pulseduration that ranges from 1 μs to 100 μs.

The light source may be any convenient light source and may includelaser and non-laser light sources. In certain embodiments, the lightsource is a non-laser light source, such as a narrow band light sourceemitting a particular wavelength or a narrow range of wavelengths. Insome instances, the narrow band light sources emit light having a narrowrange of wavelengths, such as for example, 50 nm or less, such as 40 nmor less, such as 30 nm or less, such as 25 nm or less, such as 20 nm orless, such as 15 nm or less, such as 10 nm or less, such as 5 nm orless, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Any convenientnarrow band light source protocol may be employed, such as a narrowwavelength LED.

In other embodiments, the light source is a broadband light source, suchas a broadband light source coupled to one or more optical bandpassfilters, diffraction gratings, monochromators or any combinationthereof. In some instances, the broadband light source emits lighthaving a broad range of wavelengths, such as for example, spanning 50 nmor more, such as 100 nm or more, such as 150 nm or more, such as 200 nmor more, such as 250 nm or more, such as 300 nm or more, such as 350 nmor more, such as 400 nm or more and including spanning 500 nm or more.For example, one suitable broadband light source emits light havingwavelengths from 200 nm to 1500 nm. Another example of a suitablebroadband light source includes a light source that emits light havingwavelengths from 400 nm to 1000 nm. Any convenient broadband lightsource protocol may be employed, such as a halogen lamp, deuterium arclamp, xenon arc lamp, stabilized fiber-coupled broadband light source, abroadband LED with continuous spectrum, superluminescent emitting diode,semiconductor light emitting diode, wide spectrum LED white lightsource, an multi-LED integrated white light source, among otherbroadband light sources or any combination thereof. In certainembodiments, light sources for illuminating the flow stream through theopening in the particle sorting module during image capture include anarray of infra-red LEDs.

In certain embodiments, the light source is a laser, such as acontinuous wave laser. For example, the laser may be a diode laser, suchas an ultraviolet diode laser, a visible diode laser and a near-infrareddiode laser. In other embodiments, the laser may be a helium-neon (HeNe)laser. In some instances, the laser is a gas laser, such as ahelium-neon laser, argon laser, krypton laser, xenon laser, nitrogenlaser, CO₂ laser, CO laser, argon-fluorine (ArF) excimer laser,krypton-fluorine (KrF) excimer laser, xenon chlorine (XeCl) excimerlaser or xenon-fluorine (XeF) excimer laser or a combination thereof. Inother instances, the subject systems include a dye laser, such as astilbene, coumarin or rhodamine laser. In yet other instances, lasers ofinterest include a metal-vapor laser, such as a helium-cadmium (HeCd)laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof. In still otherinstances, the light source is a solid-state laser, such as a rubylaser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser,Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphirelaser, thulim YAG laser, ytterbium YAG laser, ytterbium₂O₃ laser orcerium doped lasers and combinations thereof.

In some embodiments, the light source is a narrow bandwidth lightsource. In some instance, the light source is a light source thatoutputs a specific wavelength from 200 nm to 1500 nm, such as from 250nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to900 nm and including from 400 nm to 800 nm. In certain embodiments, thecontinuous wave light source emits light having a wavelength of 365 nm,385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm,740 nm, 770 nm or 850 nm.

The photodetector may be irradiated by the light source from anysuitable distance from, such as at a distance of 0.001 mm or more fromthe flow stream, such as 0.005 mm or more, such as 0.01 mm or more, suchas 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, suchas 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25mm or more and including at a distance of 100 mm or more. In addition,irradiation of the photodetector may be at any suitable angle such as atan angle ranging from 10° to 90°, such as from 15° to 85°, such as from20° to 80°, such as from 25° to 75° and including from 30° to 60°, forexample at a 90° angle.

In embodiments, methods include irradiating a photodetector with thelight source for two or more discrete time intervals. The term “discretetime interval” is used herein in its conventional sense to refer toirradiating the photodetector with the light source for a predeterminedduration of time followed by a period of time where the intensity of thelight source is changed (e.g., increased) and followed by a subsequentdiscrete time interval or irradiation. In some embodiments, methodsinclude irradiating the photodetector in discrete time intervals of 0.1ms or more, such as for 0.5 ms or more, such as for 1.0 ms or more, suchas for 5 ms or more, such as for 10 ms or more, such as for 20 ms ormore, such as for 30 ms or more, such as for 40 ms or more, such as for50 ms or more, such as for 60 ms or more, such as for 70 ms or more,such as for 80 ms or more, such as for 90 ms or more and including for100 ms or more. In certain embodiments, each predetermined time intervalfor irradiating the photodetector is the same duration. For instance,each predetermined time interval according to the subject methods may be50 ms. In other embodiments, each predetermined time interval isdifferent. In certain embodiments, methods include irradiating thephotodetector with the continuous wave light source at a plurality ofintensities each over a plurality of discrete time intervals, such as 3or more discrete time intervals, such as 4 or more, such as 5 or more,such as 6 or more, such as 7 or more, such as 8 or more, such as 9 ormore, such as 10 or more, such as 15 or more, such as 20 or more, suchas 25 or more, such as 50 or more, such as 75 or more and including 100or more discrete time intervals.

In some embodiments, each of the plurality of time intervals are thesame duration. In other embodiments, each of the plurality of timeintervals are a different duration. In still other embodiments, some ofthe time intervals may be the same duration and some of the timeintervals may be a different duration.

In some embodiments, the intensity of irradiation by the light source issubstantially constant for the duration of each predetermined timeinterval, such as where the intensity of irradiation varies by 10% orless, such as by 9% or less, such as by 8% or less, such as by 7% orless, such as by 6% or less, such as by 5% or less, such as by 4% orless, such as by 3% or less, such as by 2% or less, such as by 1% orless, such as by 0.5% or less, such as by 0.1% or less, such as by 0.01%or less, such as by 0.001% or less, such as by 0.0001% or less, such asby 0.00001% or less and including where the intensity of irradiation bythe light source varies by 0.000001% or less for the duration of thepredetermined time interval.

In some embodiments, the intensity of the light source is changed aftereach discrete irradiation interval. In some embodiments, the intensityof irradiation by the light source is increased. In other embodiments,the intensity of the light source is decreased. The intensity of lightused to irradiate the photodetector may be changed by 5% or more foreach subsequent time interval, such as by 10% or more, such as by 25% ormore, such as by 50% or more, such as by 75% or more, such as by 90% ormore and including by 100% or more. In certain instances, the intensityof light is changed by 1.5-fold or more, such as by 2-fold or more, suchas by 3-fold or more, such as by 4-fold or more and including by 5-foldor more. In some embodiments, methods include increasing the lightintensity for each subsequent time interval, such as by increasing thelight intensity by 5% or more for each subsequent time interval, such asby 10% or more, such as by 25% or more, such as by 50% or more, such asby 75% or more, such as by 90% or more and including by 100% or more. Inother embodiments, methods include increasing the light intensity foreach subsequent time interval by 1.5-fold or more, such as by 2-fold ormore, such as by 3-fold or more, such as by 4-fold or more and includingby 5-fold or more.

In some instances, methods include maintaining irradiation of thephotodetector by the light source while the intensity is being changed(e.g., while the light intensity is being increased). In some instances,methods include increasing the intensity of light from the light sourceover a period of time that includes at least the first predeterminedinterval and the second predetermined interval. In some instances, theintensity of light from the light source is linearly increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval. In other instances, the intensityof light from the light source is exponentially increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval.

In other instances, methods include stopping irradiation of thephotodetector by the light source for the duration the intensity of thelight source is being changed (e.g., by turning off the light source orby blocking the light source such as with a chopper, beam stop, etc.).Any convenient protocol can be used to provide intermittent irradiation,such as an electronic switch for turning the light source on-and-off,such as a switch that is computer-controlled and triggered based on adata signal (e.g., received or inputted data signal) as described ingreater detail below. In some embodiments, the time interval forchanging the intensity of the light source may be 0.001 ms or more, suchas 0.005 ms or more, such as 0.01 ms or more, such as 0.05 ms or more,such as 0.1 ms or more, such as 0.5 ms or more, such as 1 ms or more,such as 2 ms or more, such as 3 ms or more, such as 4 ms or more, suchas 5 ms or more, such as 6 ms or more, such as 7 ms or more, such as 8ms or more, such as 9 ms or more and including 10 ms or more. Forexample, the time period between each predetermined time interval forirradiating the photodetector with the light source may be from 0.001 msto 25 ms, such as from 0.005 ms to 20 ms, such as from 0.01 ms to 15 ms,such as from 0.05 ms to 10 ms and including from 0.1 ms to 5 ms.

Methods of the present disclosure, according to certain embodiments,also include detecting light with the photodetector. Photodetectors forpracticing the subject methods may be any convenient light detectingprotocol, including but not limited to photosensors or photodetectors,such as active-pixel sensors (APSs), quadrant photodiodes, imagesensors, charge-coupled devices (CCDs), intensified charge-coupleddevices (ICCDs), light emitting diodes, photon counters, bolometers,pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes,photomultiplier tubes, phototransistors, quantum dot photoconductors oravalanche photodiodes (APDs), silicon photomultiplier tubes, andcombinations thereof, among other photodetectors. In certainembodiments, the photodetector is a photomultiplier tube, such as aphotomultiplier tube having an active detecting surface area of eachregion that ranges from 0.01 cm² to 10 cm², such as from 0.05 cm² to 9cm², such as from, such as from 0.1 cm² to 8 cm², such as from 0.5 cm²to 7 cm² and including from 1 cm² to 5 cm². In other embodiments, thephotodetector is an avalanche photodiode, such as an avalanchephotodiode an active detecting surface area of each region that rangesfrom 0.01 cm² to 10 cm², such as from 0.05 cm² to 9 cm², such as from,such as from 0.1 cm² to 8 cm², such as from 0.5 cm² to 7 cm² andincluding from 1 cm² to 5 cm². In some instances, light is detected byan array of photodetectors, such as a photodetector array having 2photodetectors or more, such as 3 photodetectors or more, such as 5photodetectors or more, such as 10 photodetectors or more, such as 25photodetectors or more, such as 50 photodetectors or more, such as 75photodetectors or more, such as 100 photodetectors or more, such as 500photodetectors or more and including a photodetector array having 1000photodetectors or more. In certain instances, light is detected by anarray of avalanche photodiodes such as a photodetector array having 2avalanche photodiodes or more, such as 3 avalanche photodiodes or more,such as 5 avalanche photodiodes or more, such as 10 avalanchephotodiodes or more, such as 25 avalanche photodiodes or more, such as50 avalanche photodiodes or more, such as 75 avalanche photodiodes ormore, such as 100 avalanche photodiodes or more, such as 500 avalanchephotodiodes or more and including a photodetector array having 1000avalanche photodiodes or more.

In embodiments of the present disclosure, light may be measured by thephotodetector at one or more wavelengths, such as at 2 or morewavelengths, such as at 5 or more different wavelengths, such as at 10or more different wavelengths, such as at 25 or more differentwavelengths, such as at 50 or more different wavelengths, such as at 100or more different wavelengths, such as at 200 or more differentwavelengths, such as at 300 or more different wavelengths and includingmeasuring light from particles in the flow stream at 400 or moredifferent wavelengths.

In embodiments, light may be measured continuously or in discreteintervals. In some instances, detectors of interest are configured totake measurements of the light continuously. In other instances,detectors of interest are configured to take measurements in discreteintervals, such as measuring light every 0.001 millisecond, every 0.01millisecond, every 0.1 millisecond, every 1 millisecond, every 10milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

Measurements of the light from the light source may be taken one or moretimes during each discrete time interval, such as 2 or more times, suchas 3 or more times, such as 5 or more times and including 10 or moretimes. In certain embodiments, the light from the light source ismeasured by the photodetector 2 or more times, with the data in certaininstances being averaged.

FIGS. 1A and 1B depict the measurement of changing light intensity froma light source over a plurality discrete time intervals by aphotodetector according to certain embodiments. The photodetector isirradiated with a constant light intensity with the light source foreach 50 ms time interval followed by a 2 dB increase in light intensityover a 1 ms ramp-up time interval. FIG. 1A depicts a 50-step lightintensity ramp spanning 2601 ms. FIG. 1B depicts the first 5-steps(spanning 250 ms) of the 50-step light intensity ramp of FIG. 1A. FIG. 2depicts the measurement of light intensity that changes continuouslyaccording to certain embodiments. The photodetector is irradiated with alight intensity which increases continuously over time and data signalsare integrated over two or more predetermined time intervals todetermine the parameters of the photodetector.

FIG. 3 depicts a flow chart for determining one or more parameters of aphotodetector according to certain embodiments. At step 301, aphotodetector is irradiated with a continuous wave light source at afirst light intensity for a first predetermined discrete time interval.At step 302, the photodetector is irradiated with the light source at asecond intensity for a second predetermined discrete time interval. Atstep 303, the data signals from the photodetector are integrated over atime period that includes at least the first time interval and thesecond time interval. Based on the integrated data signals, a signalamplitude from the photodetector is calculated at step 304. One or moreparameters are determined at step 305 using the calculated signalamplitude, such as where the calculated signal amplitude is comparedwith the intensity of light during each irradiation interval. Forexample, using the comparison of the calculated signal amplitude withthe light intensity during each irradiation interval, minimum detectionthreshold 305 a, maximal detection threshold 305 b, detector sensitivity305 c, detector dynamic range 305 d, detector signal-to-noise ratio 305e or number of photoelectrons per unit output 305 f can be determined.

In embodiments, methods include integrating data signals from thephotodetector. In some embodiments, integrating the data signals fromthe photodetector includes integrating the data signals over 10% or moreof the duration of each discrete interval of irradiation, such as 15% ormore, such as 20% or more, such as 25% or more, such as 50% or more,such as 75% or more, such as 90% or more and including integrating thedata signals over 99% of the duration of each discrete interval ofirradiation. In some embodiments, data signals from the photodetectorare integrated over the entire duration of each discrete time intervalof irradiation according to the subject methods.

In some embodiments, methods include integrating data signals from thephotodetector for a period of time that includes at least each of thediscrete time intervals of irradiation at each different lightintensity. For example, where the photodetector is irradiated by thecontinuous wave light source over 50 or more discrete intervals, methodsinclude integrating data signals from the photodetector for a period oftime that includes at least the duration of the 50 discrete timeintervals. In some embodiments, methods include integrating data signalsfrom the photodetector for a period of time that includes a durationbefore irradiation of the photodetector according to the subjectmethods, such as to measure a noise component to the photodetectorsignal. In these embodiments, methods include integrating data signalsfrom the photodetector for 0.001 ms or more before irradiation of thephotodetector, such as for 0.005 ms or more, such as for 0.01 ms ormore, such as for 0.05 ms or more, such as for 0.1 ms or more, such asfor 0.5 ms or more, such as for 1 ms or more, such as for 2 ms or more,such as for 3 ms or more, such as for 4 ms or more, such as for 5 ms ormore, such as for 10 ms or more, such as for 25 ms or more, such as for50 ms or more, such as for 100 ms or more and including for 250 ms ormore before irradiation of the photodetector. In other embodiments,methods include integrating data signals from the photodetector afterthe last discrete time interval of irradiation, such as for 0.005 ms ormore, such as for 0.01 ms or more, such as for 0.05 ms or more, such asfor 0.1 ms or more, such as for 0.5 ms or more, such as for 1 ms ormore, such as for 2 ms or more, such as for 3 ms or more, such as for 4ms or more, such as for 5 ms or more, such as for 10 ms or more, such asfor 25 ms or more, such as for 50 ms or more, such as for 100 ms or moreand including for 250 ms or more.

Integrating data signals from the photodetector in certain embodimentsincludes calculating a signal amplitude over the period of time. In someinstances, calculating the signal amplitude includes calculating themedian signal amplitude. In certain instances, methods also includecomparing the calculated signal amplitude with the light intensity ofthe light source. In other instances, the methods include calculatingthe mean signal amplitude. In some instances, methods include alsocalculating the standard deviation of the signal amplitude. In otherinstances, methods include calculating the variance and coefficient ofvariation (e.g., CV=standard deviation/mean) of the signal amplitude.Based on one or more of the calculated signal amplitude and thecomparison between the calculated signal amplitude with the lightintensity of the light source, a parameter of the photodetector iscalculated. For instance, methods may include determining for thephotodetector a parameter such as minimum detection threshold, maximaldetection threshold, detector sensitivity (i.e., ratio of detectoroutput to detector input), detector dynamic range (range of detectorsignal from minimum to maximal detection thresholds), detectorsignal-to-noise ratio or number of photoelectrons per unit output.

Each of the detector parameters may be determined over a range ofoperating voltages of the photodetector. In some embodiments, theparameter is determined based on the calculated signal amplitude over10% or more of the operating voltages of the photodetector, such as 15%or more, such as 20% or more, such as 25% or more, such as 50% or more,such as 75% or more, such as 90% or more and determining the parameterover 99% or more of the operating voltages of the photodetector. Incertain instances, each of the parameters may be determined over theentire operating voltage range of the photodetector.

In certain instances, parameters of the photodetector may be adjustedbased on one or more of the calculated signal amplitude or comparisonbetween the calculated signal amplitude and the intensity of irradiationduring each discrete time interval. The term “adjusting” is used hereinin its conventional sense to refer to changing one or more functionalparameters of the photodetector. For example, adjusting thephotodetector may include increasing or decreasing a photodetectorvoltage gain. In certain embodiments, adjusting one or more parametersof the photodetector based on the calculated signal amplitude orcomparison between the calculated signal amplitude and the intensity ofirradiation during each discrete time interval of interest may be fullyautomated, such that the adjustments made require little to no humanintervention or manual input by the user.

In some embodiments, methods include determining one or more parametersof a photodetector (e.g., a photodetector in a particle analyzer) byirradiating particles in a flow stream where the particles include oneor more fluorophores. In some instances, the particles are beads (e.g.,polystyrene beads), as described in greater detail below. In someinstances, the subject methods as described below provide fordetermining parameters of the photodetector that include assignedrelative fluorescence unit (e.g., an ABD unit) per photodetector, therobust coefficient of variation (rCV) for one or more of thephotodetectors, maximum and minimum linearity per photodetector,relative change in rCV from baseline, relative change in detector gainfrom baseline and imaging specifications of the photodetector such as RFpower or axial light loss.

In some instances, methods for determining a parameter of aphotodetector include irradiating a flow stream having particles thatinclude one or more fluorophores at a first intensity for a firstpredetermined time interval and at a second intensity for a secondpredetermined time interval, detecting light from the flow stream withthe photodetector with a light source, generating a data signal from thephotodetector at the first irradiation intensity and generating a datasignal from the photodetector at the second irradiation intensity anddetermining one or more parameters of the photodetector based on thedata signals generated at the first intensity and the second intensity.

In some embodiments, methods include determining the mean fluorescenceintensity (M) from the particles at the first irradiation intensity andat the second irradiation intensity. In some instances, methods includedetermining the variance of the mean fluorescence intensity (V(M)) atthe first irradiation intensity and at the second irradiation intensity.In certain instances, methods include determining the % rCV (robustcoefficient of variation) of the photodetector. In certain embodiments,a linear fit of the variance is calculated according to:

${V(M)} = {{{c_{1}M} + c_{0}} = {{\frac{1}{Q_{led}}M} + \frac{B_{led}}{Q_{led}^{2}}}}$

where Q_(led) is given by 1/c₁ and is the statistical photo electronsper unit mean fluorescence intensity (M) (i.e., SPE/MFI). In certainembodiments, the variance is plotted to determine the linear fit of thevariance according to:

y=c ₁ x+c ₀

In embodiments, the mean fluorescence intensity and variance may bedetermined for a plurality of different irradiation intensities, such as2 or more irradiation intensities, such as 3 or more, such as 4 or more,such as 5 or more, such as 6 or more, such as 7 or more, such as 8 ormore, such as 9 or more, such as 10 or more and including 15 or moredifferent irradiation intensities.

In some embodiments, methods include determining the statistical photoelectrons (SPE) at one or more of the irradiation intensities, such asat least at the first irradiation intensity and the second irradiationintensity. In certain instances, methods further include calculatingdetector efficiency (Q_(det)) of the photodetector for each particlebased on the statistical photo electrons and the determined meanfluorescence intensity of the particle. In certain embodiments, methodsinclude determining the detector efficiency for one or detector channelof the photodetector, such as 2 or more, such as 3 or more, such as 4 ormore, such as 5 or more, such as 6 or more, such as 7 or more, such as 8or more, such as 9 or more, such as 10 or more, such as 12 or more, suchas 16 or more, such as 20 or more, such as 24 or more, such as 36 ormore, such as 48 or more, such as 72 or more and including determiningthe detector efficiency for 96 or more detector channels of thephotodetector based on the statistical photo electrons and thedetermined mean fluorescence intensity of the particle. In certaininstances, methods include determining the detector efficiency for alldetector channels of the photodetector for each particle based on thestatistical photo electrons and the determined mean fluorescenceintensity of each particle. In certain embodiments, the detectorefficiency for the photodetector is determined according to:

$Q_{Sys} = {{\frac{S\; P\; E}{M\; F\; I} \times \frac{M\; F\; I}{A\; B\; D}} = \frac{S\; P\; E}{A\; B\; D}}$

where SPE is the statistical photo electrons, MFI is the meanfluorescence intensity and ABD are assigned units per channel perparticle lot.

In certain embodiments, methods further include determining a backgroundsignal for one or more of the photodetectors. In some instances, thebackground signal is determined at one or more irradiation intensity,such as 2 or more, such as 3 or more, such as 4 or more, such as 5 ormore, such as 6 or more, such as 7 or more, such as 8 or more, such as 9or more, such as 10 or more and including 10 or more differentirradiation intensities. In some instances, the background signal isdetermined at all of the applied irradiation intensities. The backgroundsignal can likewise be determined in one or more detector channels ofthe photodetector, such as in 2 or more, such as 3 or more, such as 4 ormore, such as 5 or more, such as 6 or more, such as 7 or more, such as 8or more, such as 9 or more, such as 10 or more, such as 12 or more, suchas 16 or more, such as 20 or more, such as 24 or more, such as 36 ormore, such as 48 or more, such as 72 or more and including determiningthe background signal in 96 or more detector channels of thephotodetector, where in certain instances, the background signal isdetermined in all of the detector channels of the photodetector. In someinstances, the background signal is determined based on the statisticalphoto electrons and the detector efficiency of the photodetector. Incertain instances, the background signal is determined according to:

B _(SD) =B _(SD,MFI) ×Q _(led)

B _(bgd) =B _(SD) ²

In some embodiments, methods further include determining electronicnoise for one or more of the photodetectors. In some instances, theelectronic noise of the photodetector is determined at one or moreirradiation intensities, such as 2 or more, such as 3 or more, such as 4or more, such as 5 or more, such as 6 or more, such as 7 or more, suchas 8 or more, such as 9 or more, such as 10 or more and including 10 ormore different irradiation intensities. In some instances, theelectronic noise of the photodetector is determined at all of theapplied irradiation intensities. The electronic noise can likewise bedetermined in one or more detector channels of the photodetector, suchas in 2 or more, such as 3 or more, such as 4 or more, such as 5 ormore, such as 6 or more, such as 7 or more, such as 8 or more, such as 9or more, such as 10 or more, such as 12 or more, such as 16 or more,such as 20 or more, such as 24 or more, such as 36 or more, such as 48or more, such as 72 or more and including determining the electronicnoise in 96 or more detector channels of the photodetector, where incertain instances, the electronic noise is determined in all of thedetector channels of the photodetector. In some instances, theelectronic noise is determined based on the statistical photo electronsand the detector efficiency of the photodetector. In certain instances,the electronic noise is determined according to:

EN_(SD)=EN_(SD,MFI) ×Q _(led)

EN=EN_(SD) ²

In some embodiments, methods further include determining the limit ofdetection of one or more of the photodetectors. In some instances, thelimit of detection of the photodetector is determined in one or moredetector channels of the photodetector, such as in 2 or more, such as 3or more, such as 4 or more, such as 5 or more, such as 6 or more, suchas 7 or more, such as 8 or more, such as 9 or more, such as 10 or more,such as 12 or more, such as 16 or more, such as 20 or more, such as 24or more, such as 36 or more, such as 48 or more, such as 72 or more andincluding determining the limit of detection of the photodetector in 96or more detector channels of the photodetector, where in certaininstances, the limit of detection is determined in all of the detectorchannels of the photodetector. In some instances, the limit of detectionof each photodetector is determined according to:

2+2SD=4(1+B _(SD))

In some embodiments, methods further include determining the detectorphotosensitivity of one or more photodetectors. In certain embodiments,determining the detector photosensitivity of the photodetector includessetting up an initial detector gain for the photodetector. In someinstances, methods include irradiating the photodetector with the lightsource (such as described in detail above) at a plurality of differentlight intensities, generating data signals from the photodetector forthe plurality of light intensities at one or more detector gains of thephotodetector and determining the lowest light irradiation intensitythat generates a data signal resolvable from the background data signalat each detector gain. In some instances, methods include determiningthe lowest light irradiation intensity that generates a data signal thatfalls two standard deviations from the background data signal at eachdetector gain. In certain instances, methods include setting thedetector gain to the gain where the lowest light irradiation intensitythat generates a data signal resolvable from the background data signalplateaus when plotted as a function of light intensity. FIG. 3B depictsa plot used for setting up an initial detector gain for a photodetectoraccording to certain embodiments. As shown in FIG. 3B, detector gain ofthe photodetector is plotted as a function of light (e.g., LED)irradiation intensity for two different fluorophores (e.g., fluorophoresstably associated with a particle as described in greater detail below)In setting up an initial detector gain for the photodetector, thedetector gain is determined where the lowest light irradiation intensitythat generates a data signal resolvable from the background data signalplateaus, which in FIG. 3B is about 575 volts.

Systems for Determining a Parameter of a Photodetector

Aspects of the present disclosure also include systems having a lightsource and a photodetector that is configured to detect light from thelight source at different irradiation intensities for predetermined timeintervals. In some embodiments, the light source is a continuous wavelight source. The term “continuous wave light source” is used herein inits conventional sense to refer to a source of light which providesuninterrupted light flux and maintains irradiation of the photodetectorwith little to no undesired changes in light intensity. In someembodiments, the continuous light source emits non-pulsed ornon-stroboscopic irradiation. In certain embodiments, the continuouslight source provides for substantially constant emitted lightintensity. For instance, the continuous light source may provide foremitted light intensity during a time interval of irradiation thatvaries by 10% or less, such as by 9% or less, such as by 8% or less,such as by 7% or less, such as by 6% or less, such as by 5% or less,such as by 4% or less, such as by 3% or less, such as by 2% or less,such as by 1% or less, such as by 0.5% or less, such as by 0.1% or less,such as by 0.01% or less, such as by 0.001% or less, such as by 0.0001%or less, such as by 0.00001% or less and including where the emittedlight intensity during a time interval of irradiation varies by0.000001% or less.

In certain instances, the light source is a pulsed light source. Theterm “pulsed light source” is used herein in its conventional sense torefer to a source of light which provides light flux in predeterminedintervals, such as by stroboscopic irradiation. The pulse duration mayvary depending on the type of the light source and may be 0.001 ns ormore, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 nsor more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns ormore, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more,such as 10 ns or more, such as 25 ns or more, such as 50 ns or more,such as 100 ns or more, such as 500 ns or more, such as 1000 ns or moreand including a pulse duration of 5 μs or more. For example, the pulseduration of pulsed light sources may range from 0.00001 μs to 1000 μs,such as from 0.00005 μs to 900 μs, such as from 0.0001 μs to 800 μs,such as from 0.0005 μs to 700 μs, such as from 0.001 μs to 600 μs, suchas from 0.005 μs to 500 μs, such as from 0.01 μs to 400 μs, such as from0.05 μs to 300 μs, such as from 0.1 μs to 200 μs and including a pulseduration that ranges from 1 μs to 100 μs.

The continuous wave light source may be any convenient light source andmay include laser and non-laser light sources. In certain embodiments,the light source is a non-laser light source, such as a narrow bandlight source emitting a particular wavelength or a narrow range ofwavelengths. In some instances, the narrow band light sources emit lighthaving a narrow range of wavelengths, such as for example, 50 nm orless, such as 40 nm or less, such as 30 nm or less, such as 25 nm orless, such as 20 nm or less, such as 15 nm or less, such as 10 nm orless, such as 5 nm or less, such as 2 nm or less and including lightsources which emit a specific wavelength of light (i.e., monochromaticlight). Any convenient narrow band light source protocol may beemployed, such as a narrow wavelength LED.

In other embodiments, the light source is a broadband light source, suchas a broadband light source coupled to one or more optical bandpassfilters, diffraction gratings, monochromators or any combinationthereof. In some instances, the broadband light source emits lighthaving a broad range of wavelengths, such as for example, spanning 50 nmor more, such as 100 nm or more, such as 150 nm or more, such as 200 nmor more, such as 250 nm or more, such as 300 nm or more, such as 350 nmor more, such as 400 nm or more and including spanning 500 nm or more.For example, one suitable broadband light source emits light havingwavelengths from 200 nm to 1500 nm. Another example of a suitablebroadband light source includes a light source that emits light havingwavelengths from 400 nm to 1000 nm. Any convenient broadband lightsource protocol may be employed, such as a halogen lamp, deuterium arclamp, xenon arc lamp, stabilized fiber-coupled broadband light source, abroadband LED with continuous spectrum, superluminescent emitting diode,semiconductor light emitting diode, wide spectrum LED white lightsource, an multi-LED integrated white light source, among otherbroadband light sources or any combination thereof. In certainembodiments, light sources for illuminating the flow stream through theopening in the particle sorting module during image capture include anarray of infra-red LEDs.

In certain embodiments, the light source is a laser, such as continuouswave laser. For example, the laser may be a diode laser, such as anultraviolet diode laser, a visible diode laser and a near-infrared diodelaser. In other embodiments, the laser may be a helium-neon (HeNe)laser. In some instances, the laser is a gas laser, such as ahelium-neon laser, argon laser, krypton laser, xenon laser, nitrogenlaser, CO₂ laser, CO laser, argon-fluorine (ArF) excimer laser,krypton-fluorine (KrF) excimer laser, xenon chlorine (XeCl) excimerlaser or xenon-fluorine (XeF) excimer laser or a combination thereof. Inother instances, the subject systems include a dye laser, such as astilbene, coumarin or rhodamine laser. In yet other instances, lasers ofinterest include a metal-vapor laser, such as a helium-cadmium (HeCd)laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof. In still otherinstances, the subject systems include a solid-state laser, such as aruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser,Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphirelaser, thulim YAG laser, ytterbium YAG laser, ytterbium₂O₃ laser orcerium doped lasers and combinations thereof.

In some embodiments, the light source is a narrow bandwidth lightsource. In some instance, the light source is a light source thatoutputs a specific wavelength from 200 nm to 1500 nm, such as from 250nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to900 nm and including from 400 nm to 800 nm. In certain embodiments, thelight source emits light having a wavelength of 365 nm, 385 nm, 405 nm,460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nmor 850 nm. In certain embodiments, the wavelength of light outputted bythe light source is matched with an optical adjustment component usedwhen irradiating the photodetector such as a bandpass filter or dichroicmirror. In some instances, wavelength of light outputted by the lightsource is matched with the spectral bandwidth of a bandpass filter inoptical communication with the photodetector.

The light source may be positioned any suitable distance from thephotodetector, such as where the light source and the photodetector areseparated by 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mmor more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm ormore, such as 5 mm or more, such as 10 mm or more, such as 25 mm or moreand including at a distance of 100 mm or more. In addition, the lightsource may be positioned at any suitable angle to the photodetector,such as at an angle ranging from 10° to 90°, such as from 15° to 85°,such as from 20° to 80°, such as from 25° to 75° and including from 30°to 60°, for example at a 90° angle.

Light sources according to certain embodiments may also include one ormore optical adjustment components. The term “optical adjustment” isused herein in its conventional sense to refer to any device that iscapable of changing the spatial width of irradiation or some othercharacteristic of irradiation from the light source, such as forexample, irradiation direction, wavelength, beam width, beam intensityand focal spot. Optical adjustment protocols may be any convenientdevice which adjusts one or more characteristics of the light source,including but not limited to lenses, mirrors, filters, fiber optics,wavelength separators, pinholes, slits, collimating protocols andcombinations thereof. In certain embodiments, systems of interestinclude one or more focusing lenses. The focusing lens, in one examplemay be a de-magnifying lens. In another example, the focusing lens is amagnifying lens. In other embodiments, systems of interest include oneor more mirrors. In still other embodiments, systems of interest includefiber optics.

Where the optical adjustment component is configured to move, theoptical adjustment component may be configured to be moved continuouslyor in discrete intervals. In some embodiments, movement of the opticaladjustment component is continuous. In other embodiments, the opticaladjustment component is movable in discrete intervals, such as forexample in 0.01 micron or greater increments, such as 0.05 micron orgreater, such as 0.1 micron or greater, such as 0.5 micron or greater,such as 1 micron or greater, such as 10 micron or greater, such as 100microns or greater, such as 500 microns or greater, such as 1 mm orgreater, such as 5 mm or greater, such as 10 mm or greater and including25 mm or greater increments.

Any displacement protocol may be employed to move the optical adjustmentcomponent structures, such as coupled to a movable support stage ordirectly with a motor actuated translation stage, leadscrew translationassembly, geared translation device, such as those employing a steppermotor, servo motor, brushless electric motor, brushed DC motor,micro-step drive motor, high resolution stepper motor, among other typesof motors.

In embodiments, the continuous wave light source is configured forirradiating for two or more discrete time intervals, each time intervalbeing at a different irradiation intensity. In some embodiments, thecontinuous wave light source is configured for irradiation at aparticular intensity for time intervals of 0.1 ms or more, such as for0.5 ms or more, such as for 1.0 ms or more, such as for 5 ms or more,such as for 10 ms or more, such as for 20 ms or more, such as for 30 msor more, such as for 40 ms or more, such as for 50 ms or more, such asfor 60 ms or more, such as for 70 ms or more, such as for 80 ms or more,such as for 90 ms or more and including for 100 ms or more. Forinstance, the continuous wave light source may be configured forirradiation at a particular light intensity of 50 ms.

In some embodiments, the light source is configured to maintain asubstantially constant light intensity for the duration of eachpredetermined time interval, such as where the intensity of irradiationvaries by 10% or less, such as by 9% or less, such as by 8% or less,such as by 7% or less, such as by 6% or less, such as by 5% or less,such as by 4% or less, such as by 3% or less, such as by 2% or less,such as by 1% or less, such as by 0.5% or less, such as by 0.1% or less,such as by 0.01% or less, such as by 0.001% or less, such as by 0.0001%or less, such as by 0.00001% or less and including where the intensityof irradiation by the light source varies by 0.000001% or less for theduration of the predetermined time interval.

In some instances, the light source is configured to increase intensityover a period of time that includes at least the first predeterminedinterval and the second predetermined interval. In some instances, theintensity of light from the light source is linearly increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval. In other instances, the intensityof light from the light source is exponentially increased over theperiod of time that includes at least the first predetermined intervaland the second predetermined interval.

Photodetectors of the subject systems may be any convenient lightdetecting protocol, including but not limited to photosensors orphotodetectors, such as active-pixel sensors (APSs), quadrantphotodiodes, image sensors, charge-coupled devices (CCDs), intensifiedcharge-coupled devices (ICCDs), light emitting diodes, photon counters,bolometers, pyroelectric detectors, photoresistors, photovoltaic cells,photodiodes, photomultiplier tubes, phototransistors, quantum dotphotoconductors or avalanche photodiodes, silicon photomultiplier tubesand combinations thereof, among other photodetectors. In certainembodiments, the photodetector is a photomultiplier tube, such as aphotomultiplier tube having an active detecting surface area of eachregion that ranges from 0.01 cm² to 10 cm², such as from 0.05 cm² to 9cm², such as from, such as from 0.1 cm² to 8 cm², such as from 0.5 cm²to 7 cm² and including from 1 cm² to 5 cm². In other embodiments, thephotodetector is an avalanche photodiode, such as an avalanchephotodiode an active detecting surface area of each region that rangesfrom 0.01 cm² to 10 cm², such as from 0.05 cm² to 9 cm², such as from,such as from 0.1 cm² to 8 cm², such as from 0.5 cm² to 7 cm² andincluding from 1 cm² to 5 cm². In some instances, light is detected byan array of photodetectors, such as a photodetector array having 2photodetectors or more, such as 3 photodetectors or more, such as 5photodetectors or more, such as 10 photodetectors or more, such as 25photodetectors or more, such as 50 photodetectors or more, such as 75photodetectors or more, such as 100 photodetectors or more, such as 500photodetectors or more and including a photodetector array having 1000photodetectors or more. In certain instances, light is detected by anarray of avalanche photodiodes such as a photodetector array having 2avalanche photodiodes or more, such as 3 avalanche photodiodes or more,such as 5 avalanche photodiodes or more, such as 10 avalanchephotodiodes or more, such as 25 avalanche photodiodes or more, such as50 avalanche photodiodes or more, such as 75 avalanche photodiodes ormore, such as 100 avalanche photodiodes or more, such as 500 avalanchephotodiodes or more and including a photodetector array having 1000avalanche photodiodes or more.

In embodiments of the present disclosure, the photodetector may beconfigured to detect light at one or more wavelengths, such as at 2 ormore wavelengths, such as at 5 or more different wavelengths, such as at10 or more different wavelengths, such as at 25 or more differentwavelengths, such as at 50 or more different wavelengths, such as at 100or more different wavelengths, such as at 200 or more differentwavelengths, such as at 300 or more different wavelengths and includingmeasuring light from particles in the flow stream at 400 or moredifferent wavelengths.

In embodiments, photodetectors may be configured to measure lightcontinuously or in discrete intervals. In some instances, detectors ofinterest are configured to take measurements of the light continuously.In other instances, detectors of interest are configured to takemeasurements in discrete intervals, such as measuring light every 0.001millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1millisecond, every 10 milliseconds, every 100 milliseconds and includingevery 1000 milliseconds, or some other interval.

Measurements of the light from the light source may be taken one or moretimes during each discrete time interval, such as 2 or more times, suchas 3 or more times, such as 5 or more times and including 10 or moretimes. In certain embodiments, the light from the light source ismeasured by the photodetector 2 or more times, with the data in certaininstances being averaged.

In embodiments, systems also include a processor having memory operablycoupled to the processor wherein the memory includes instructions storedthereon, which when executed by the processor, cause the processor tointegrate data signals from the photodetector over a period of time thatincludes each of the discrete irradiation intervals and determine one ormore parameters of the photodetector based on the integrated datasignals. In certain embodiments, the memory includes instructions whichwhen executed by the processor, cause the processor to calculate themedian signal amplitude. In other instances, the memory includesinstructions which when executed by the processor, cause the processorto calculate the mean signal amplitude. In some instances, the memoryincludes instructions which when executed by the processor, cause theprocessor to also calculate the standard deviation of the signalamplitude. In other instances, the memory includes instructions whichwhen executed by the processor, cause the processor to also calculatethe variance and coefficient of variation (e.g., CV=standarddeviation/mean) of the signal amplitude In certain embodiments, systemsinclude a processor having memory operably coupled to the processorwherein the memory includes instructions stored thereon, which whenexecuted by the processor, cause the processor to compare the calculatedsignal amplitude with the light intensity of the light source.

In some embodiments, systems include a processor having memory operablycoupled to the processor wherein the memory includes instructions storedthereon, which when executed by the processor, cause the processor tocompare the calculated signal amplitude with the light intensity of thelight source. Based on one or more of the calculated signal amplitudeand the comparison between the calculated signal amplitude with thelight intensity of the light source, systems of interest include memoryhaving instructions for calculating a parameter of the photodetector.For instance, the memory may include instructions for determining forthe photodetector a parameter such as minimum detection threshold,maximal detection threshold, detector sensitivity (i.e., ratio ofdetector output to detector input), detector dynamic range (range ofdetector signal from minimum to maximal detection thresholds), detectorsignal-to-noise ratio or number of photoelectrons per unit output. Insome embodiments, the memory includes instructions for determining thephotodetector parameters over a range of the operating voltages of thephotodetector. In certain embodiments, the memory includes instructionsfor calculating signal amplitude of the photodetector over 10% or moreof the operating voltages of the photodetector, such as 15% or more,such as 20% or more, such as 25% or more, such as 50% or more, such as75% or more, such as 90% or more and determining the parameter over 99%or more of the operating voltages of the photodetector. In certaininstances, the memory includes instructions for calculating signalamplitude of the photodetector over the entire operating voltage rangeof the photodetector.

In some embodiments, systems include a processor having memory operablycoupled to the processor wherein the memory includes instructions storedthereon, which when executed by the processor, cause the processor todetermine one or more parameters of a photodetector where the memoryincludes instructions to irradiate particles in a flow stream where theparticles (e.g., multispectral beads as described below) include one ormore fluorophores. In some instances, the memory includes instructionswhich when executed by the processor cause the processor to determineparameters of the photodetector that include assigned relativefluorescence unit (e.g., an ABD unit) per photodetector, the robustcoefficient of variation (rCV) for one or more of the photodetectors,maximum and minimum linearity per photodetector, relative change in rCVfrom baseline, relative change in detector gain from baseline andimaging specifications of the photodetector such as RF power or axiallight loss.

In some instances, the memory includes instructions for determining aparameter of a photodetector includes irradiating a flow stream havingparticles that include one or more fluorophores at a first intensity fora first predetermined time interval and at a second intensity for asecond predetermined time interval, detecting light from the flow streamwith the photodetector with a light source, generating a data signalfrom the photodetector at the first irradiation intensity and generatinga data signal from the photodetector at the second irradiation intensityand determining one or more parameters of the photodetector based on thedata signals generated at the first intensity and the second intensity.

In some embodiments, the memory includes instructions for determiningthe mean fluorescence intensity (M) from the particles at the firstirradiation intensity and at the second irradiation intensity. In someinstances, the memory includes instructions for determining the varianceof the mean fluorescence intensity (V(M)) at the first irradiationintensity and at the second irradiation intensity. In certain instances,the memory includes instructions for determining the % rCV (robustcoefficient of variation) of the photodetector. In certain embodiments,the memory includes instructions which when executed by the processorcause the process to calculate a linear fit of the variance accordingto:

${V(M)} = {{{c_{1}M} + c_{0}} = {{\frac{1}{Q_{led}}M} + \frac{B_{led}}{Q_{led}^{2}}}}$

where (led is given by 1/c₁ and is the statistical photo electrons perunit mean fluorescence intensity (M) (i.e., SPE/MFI). In certainembodiments, the memory includes instructions for plotting variance inorder to determine the linear fit of the variance according to:

y=c ₁ x+c ₀

In embodiments, the mean fluorescence intensity and variance may bedetermined for a plurality of different irradiation intensities, such as2 or more irradiation intensities, such as 3 or more, such as 4 or more,such as 5 or more, such as 6 or more, such as 7 or more, such as 8 ormore, such as 9 or more, such as 10 or more and including 15 or moredifferent irradiation intensities.

In some embodiments, the memory includes instructions for determiningthe statistical photo electrons (SPE) at one or more of the irradiationintensities, such as at least at the first irradiation intensity and thesecond irradiation intensity. In certain instances, the memory includesinstructions for calculating detector efficiency (Q_(det)) of thephotodetector for each particle based on the statistical photo electronsand the determined mean fluorescence intensity of the particle. Incertain embodiments, the memory includes instructions for determiningthe detector efficiency for one or more detector channels of thephotodetector, such as 2 or more, such as 3 or more, such as 4 or more,such as 5 or more, such as 6 or more, such as 7 or more, such as 8 ormore, such as 9 or more, such as 10 or more, such as 12 or more, such as16 or more, such as 20 or more, such as 24 or more, such as 36 or more,such as 48 or more, such as 72 or more and including for determining thedetector efficiency for 96 or more detector channels of thephotodetector based on the statistical photo electrons and thedetermined mean fluorescence intensity of the particle. In certaininstances, the memory includes instructions for determining the detectorefficiency for all detector channels of the photodetector for eachparticle based on the statistical photo electrons and the determinedmean fluorescence intensity of each particle. In certain embodiments,the memory includes instructions which when executed by the processorcause the processor to determine detector efficiency determinedaccording to:

$Q_{Sys} = {{\frac{S\; P\; E}{M\; F\; I} \times \frac{M\; F\; I}{A\; B\; D}} = \frac{S\; P\; E}{A\; B\; D}}$

where SPE is the statistical photo electrons, MFI is the meanfluorescence intensity and ABD are assigned units per channel perparticle lot.

In certain embodiments, the memory includes instructions for determininga background signal for one or more of the photodetectors. In someinstances, the background signal is determined at one or moreirradiation intensities, such as 2 or more, such as 3 or more, such as 4or more, such as 5 or more, such as 6 or more, such as 7 or more, suchas 8 or more, such as 9 or more, such as 10 or more and including 10 ormore different irradiation intensities. In some instances, the memoryincludes instructions to determine background signal at all of theapplied irradiation intensities. In some embodiments, the memoryincludes instructions for determining the background signal in one ormore detector channels of the photodetector, such as in 2 or more, suchas 3 or more, such as 4 or more, such as 5 or more, such as 6 or more,such as 7 or more, such as 8 or more, such as 9 or more, such as 10 ormore, such as 12 or more, such as 16 or more, such as 20 or more, suchas 24 or more, such as 36 or more, such as 48 or more, such as 72 ormore and including where the memory includes instructions fordetermining the background signal in 96 or more detector channels of thephotodetector. In certain instances, the memory includes instructionsfor determining the background signal in all of the detector channels ofthe photodetector. In some instances, the memory includes instructionswhich when executed by the processor, cause the processor to determinethe background signal based on the statistical photo electrons and thedetector efficiency of the photodetector. In certain instances, thememory includes instructions for determining background signal accordingto:

B _(SD) =B _(SD,MFI) ×Q _(led)

B _(bgd) =B _(SD)

In some embodiments, the memory includes instructions for determiningelectronic noise for one or more of the photodetectors. In someinstances, the electronic noise of the photodetector is determined atone or more irradiation intensities, such as 2 or more, such as 3 ormore, such as 4 or more, such as 5 or more, such as 6 or more, such as 7or more, such as 8 or more, such as 9 or more, such as 10 or more andincluding 10 or more different irradiation intensities. In someinstances, the electronic noise of the photodetector is determined atall of the applied irradiation intensities. The electronic noise canlikewise be determined in one or more detector channels of thephotodetector, such as in 2 or more, such as 3 or more, such as 4 ormore, such as 5 or more, such as 6 or more, such as 7 or more, such as 8or more, such as 9 or more, such as 10 or more, such as 12 or more, suchas 16 or more, such as 20 or more, such as 24 or more, such as 36 ormore, such as 48 or more, such as 72 or more and including determiningthe electronic noise in 96 or more detector channels of thephotodetector. In certain instances, the memory includes instructionsfor determining the electronic noise in all of the detector channels ofthe photodetector. In some instances, the memory includes instructionsfor determining the electronic noise based on the statistical photoelectrons and the detector efficiency of the photodetector. In certaininstances, the memory includes instructions for determining theelectronic noise according to:

EN_(SD)=EN_(SD,MFI) ×Q _(led)

EN=EN_(SD) ²

In some embodiments, the memory includes instructions for determiningthe limit of detection of one or more of the photodetectors. In someinstances, the memory includes instructions for determining the limit ofdetection of the photodetector in one or more detector channels of thephotodetector, such as in 2 or more, such as 3 or more, such as 4 ormore, such as 5 or more, such as 6 or more, such as 7 or more, such as 8or more, such as 9 or more, such as 10 or more, such as 12 or more, suchas 16 or more, such as 20 or more, such as 24 or more, such as 36 ormore, such as 48 or more, such as 72 or more and including where thememory includes instructions for determining the limit of detection ofthe photodetector in 96 or more detector channels of the photodetector.In certain instances, the memory includes instructions for determiningthe limit of detection in all of the detector channels of thephotodetector. In some instances, the memory includes instructions fordetermining the limit of detection of each photodetector according to:

2+2SD=4(1+B _(SD))

In some embodiments, the memory includes instructions for determiningthe detector photosensitivity of one or more photodetectors. In certainembodiments, the memory includes instructions for setting up an initialdetector gain for the photodetector. In some instances, the memoryincludes instructions for irradiating the photodetector with the lightsource (as described in detail above) at a plurality of different lightintensities, instructions for generating data signals from thephotodetector for the plurality of light intensities at one or moredetector gains of the photodetector and instructions for determining thelowest light irradiation intensity that generates a data signalresolvable from the background data signal at each detector gain. Insome instances, the memory includes instructions for determining thelowest light irradiation intensity that generates a data signal thatfalls two standard deviations from the background data signal at eachdetector gain. In certain instances, the memory includes instructionsfor setting the detector gain to the gain where the lowest lightirradiation intensity that generates a data signal resolvable from thebackground data signal plateaus when plotted as a function of lightintensity.

In certain embodiments, the photodetector is a photodetector that ispositioned in a particle analyzer, such as a particle sorter. In certainembodiments, the subject systems are flow cytometric systems thatincludes the photodetector as part of a light detection system fordetecting light emitted by a sample in a flow stream. Suitable flowcytometry systems may include, but are not limited to those described inOrmerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press(1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods inMolecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry,3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem.January; 49(pt 1):17-28; Linden, et. al., Semin Throm Hemost. 2004October; 30(5):502-11; Alison, et al. J Pathol, 2010 December;222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug CarrierSyst. 24(3):203-255; the disclosures of which are incorporated herein byreference. In certain instances, flow cytometry systems of interestinclude BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flowcytometer, BD Biosciences FACSCelesta™ flow cytometer, BD BiosciencesFACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BDBiosciences FACSymphony™ flow cytometer BD Biosciences LSRFortessa™ flowcytometer, BD Biosciences LSRFortess™ X-20 flow cytometer and BDBiosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cellsorter, BD Biosciences FACSLyric™ cell sorter and BD Biosciences Via™cell sorter BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™cell sorter, BD Biosciences Aria™ cell sorters and BD BiosciencesFACSMelody™ cell sorter, or the like.

In some embodiments, the subject particle sorting systems are flowcytometric systems, such those described in U.S. Pat. Nos. 10,006,852;9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334;9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146;8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017;6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842;5,602,039; the disclosure of which are herein incorporated by referencein their entirety.

In certain embodiments, the subject systems are flow cytometric systemshaving an excitation module that uses radio-frequency multiplexedexcitation to generate a plurality of frequency shifted beams of light.In certain instances, the subject systems are flow cytometric systems asdescribed in U.S. Pat. Nos. 9,423,353 and 9,784,661 and U.S. PatentPublication Nos. 2017/0133857 and 2017/0350803, the disclosures of whichare herein incorporated by reference.

In some embodiments, the subject systems are particle sorting systemsthat are configured to sort particles with an enclosed particle sortingmodule, such as those described in U.S. Patent Publication No.2017/0299493, filed on Mar. 28, 2017, the disclosure of which isincorporated herein by reference. In certain embodiments, particles(e.g, cells) of the sample are sorted using a sort decision modulehaving a plurality of sort decision units, such as those described inU.S. Provisional Patent Application No. 62/803,264, filed on Feb. 8,2019, the disclosure of which is incorporated herein by reference. Insome embodiments, methods for sorting components of sample includesorting particles (e.g., cells in a biological sample) with a particlesorting module having deflector plates, such as described in U.S. PatentPublication No. 2017/0299493, filed on Mar. 28, 2017, the disclosure ofwhich is incorporated herein by reference.

In some embodiments, systems of interest include a particle analysissystem which can be used to analyze and characterize particles, with orwithout physically sorting the particles into collection vessels. FIG.4A shows a functional block diagram of an example of a particle analysissystem. In some embodiments, the particle analysis system 401 is a flowsystem. The particle analysis system 401 shown in FIG. 4A can beconfigured to perform, in whole or in part, the methods described hereinsuch as. The particle analysis system 401 includes a fluidics system402. The fluidics system 402 can include or be coupled with a sampletube 405 and a moving fluid column within the sample tube in whichparticles 403 (e.g. cells) of a sample move along a common sample path409.

The particle analysis system 401 includes a detection system 404configured to collect a signal from each particle as it passes one ormore detection stations along the common sample path. A detectionstation 408 generally refers to a monitored area 407 of the commonsample path. Detection can, in some implementations, include detectinglight or one or more other properties of the particles 403 as they passthrough a monitored area 407. In FIG. 4A, one detection station 408 withone monitored area 407 is shown. Some implementations of the particleanalysis system 401 can include multiple detection stations.Furthermore, some detection stations can monitor more than one area.

Each signal is assigned a signal value to form a data point for eachparticle. As described above, this data can be referred to as eventdata. The data point can be a multidimensional data point includingvalues for respective properties measured for a particle. The detectionsystem 404 is configured to collect a succession of such data points ina first time interval.

The particle analysis system 401 can also include a control system 306.The control system 406 can include one or more processors, an amplitudecontrol circuit and/or a frequency control circuit. The control systemshown can be operationally associated with the fluidics system 402. Thecontrol system can be configured to generate a calculated signalfrequency for at least a portion of the first time interval based on aPoisson distribution and the number of data points collected by thedetection system 404 during the first time interval. The control system406 can be further configured to generate an experimental signalfrequency based on the number of data points in the portion of the firsttime interval. The control system 406 can additionally compare theexperimental signal frequency with that of a calculated signal frequencyor a predetermined signal frequency.

FIG. 4B shows a system 400 for flow cytometry in accordance with anillustrative embodiment of the present invention. The system 400includes a flow cytometer 410, a controller/processor 490 and a memory495. The flow cytometer 410 includes one or more excitation lasers 415a-415 c, a focusing lens 420, a flow chamber 425, a forward scatterdetector 430, a side scatter detector 435, a fluorescence collectionlens 440, one or more beam splitters 445 a-445 g, one or more bandpassfilters 450 a-450 e, one or more longpass (“LP”) filters 455 a-455 b,and one or more fluorescent detectors 460 a-460 f.

The excitation lasers 115 a-c emit light in the form of a laser beam.The wavelengths of the laser beams emitted from excitation lasers 415a-415 c are 488 nm, 633 nm, and 325 nm, respectively, in the examplesystem of FIG. 4B. The laser beams are first directed through one ormore of beam splitters 445 a and 445 b. Beam splitter 445 a transmitslight at 488 nm and reflects light at 633 nm. Beam splitter 445 btransmits UV light (light with a wavelength in the range of 10 to 400nm) and reflects light at 488 nm and 633 nm.

The laser beams are then directed to a focusing lens 420, which focusesthe beams onto the portion of a fluid stream where particles of a sampleare located, within the flow chamber 425. The flow chamber is part of afluidics system which directs particles, typically one at a time, in astream to the focused laser beam for interrogation. The flow chamber cancomprise a flow cell in a benchtop cytometer or a nozzle tip in astream-in-air cytometer.

The light from the laser beam(s) interacts with the particles in thesample by diffraction, refraction, reflection, scattering, andabsorption with re-emission at various different wavelengths dependingon the characteristics of the particle such as its size, internalstructure, and the presence of one or more fluorescent moleculesattached to or naturally present on or in the particle. The fluorescenceemissions as well as the diffracted light, refracted light, reflectedlight, and scattered light may be routed to one or more of the forwardscatter detector 430, the side scatter detector 435, and the one or morefluorescent detectors 460 a-460 f through one or more of the beamsplitters 445 a-445 g, the bandpass filters 450 a-450 e, the longpassfilters 455 a-455 b, and the fluorescence collection lens 440.

The fluorescence collection lens 440 collects light emitted from theparticle-laser beam interaction and routes that light towards one ormore beam splitters and filters. Bandpass filters, such as bandpassfilters 450 a-450 e, allow a narrow range of wavelengths to pass throughthe filter. For example, bandpass filter 450 a is a 510/20 filter. Thefirst number represents the center of a spectral band. The second numberprovides a range of the spectral band. Thus, a 510/20 filter extends 10nm on each side of the center of the spectral band, or from 500 nm to520 nm. Shortpass filters transmit wavelengths of light equal to orshorter than a specified wavelength. Longpass filters, such as longpassfilters 455 a-455 b, transmit wavelengths of light equal to or longerthan a specified wavelength of light. For example, longpass filter 455a, which is a 670 nm longpass filter, transmits light equal to or longerthan 670 nm. Filters are often selected to optimize the specificity of adetector for a particular fluorescent dye. The filters can be configuredso that the spectral band of light transmitted to the detector is closeto the emission peak of a fluorescent dye.

Beam splitters direct light of different wavelengths in differentdirections. Beam splitters can be characterized by filter propertiessuch as shortpass and longpass. For example, beam splitter 445 g is a620 SP beam splitter, meaning that the beam splitter 445 g transmitswavelengths of light that are 620 nm or shorter and reflects wavelengthsof light that are longer than 620 nm in a different direction. In oneembodiment, the beam splitters 445 a-445 g can comprise optical mirrors,such as dichroic mirrors.

The forward scatter detector 430 is positioned slightly off axis fromthe direct beam through the flow cell and is configured to detectdiffracted light, the excitation light that travels through or aroundthe particle in mostly a forward direction. The intensity of the lightdetected by the forward scatter detector is dependent on the overallsize of the particle. The forward scatter detector can include aphotodiode. The side scatter detector 435 is configured to detectrefracted and reflected light from the surfaces and internal structuresof the particle, and tends to increase with increasing particlecomplexity of structure. The fluorescence emissions from fluorescentmolecules associated with the particle can be detected by the one ormore fluorescent detectors 460 a-460 f. The side scatter detector 435and fluorescent detectors can include photomultiplier tubes. The signalsdetected at the forward scatter detector 430, the side scatter detector435 and the fluorescent detectors can be converted to electronic signals(voltages) by the detectors. This data can provide information about thesample.

One of skill in the art will recognize that a flow cytometer inaccordance with an embodiment of the present invention is not limited tothe flow cytometer depicted in FIG. 4B, but can include any flowcytometer known in the art. For example, a flow cytometer may have anynumber of lasers, beam splitters, filters, and detectors at variouswavelengths and in various different configurations.

In operation, cytometer operation is controlled by acontroller/processor 490, and the measurement data from the detectorscan be stored in the memory 495 and processed by thecontroller/processor 490. Although not shown explicitly, thecontroller/processor 190 is coupled to the detectors to receive theoutput signals therefrom, and may also be coupled to electrical andelectromechanical components of the flow cytometer 400 to control thelasers, fluid flow parameters, and the like. Input/output (I/O)capabilities 497 may be provided also in the system. The memory 495,controller/processor 490, and I/O 497 may be entirely provided as anintegral part of the flow cytometer 410. In such an embodiment, adisplay may also form part of the I/O capabilities 497 for presentingexperimental data to users of the cytometer 400. Alternatively, some orall of the memory 495 and controller/processor 490 and I/O capabilitiesmay be part of one or more external devices such as a general purposecomputer. In some embodiments, some or all of the memory 495 andcontroller/processor 490 can be in wireless or wired communication withthe cytometer 410. The controller/processor 490 in conjunction with thememory 495 and the I/O 497 can be configured to perform variousfunctions related to the preparation and analysis of a flow cytometerexperiment.

The system illustrated in FIG. 4B includes six different detectors thatdetect fluorescent light in six different wavelength bands (which may bereferred to herein as a “filter window” for a given detector) as definedby the configuration of filters and/or splitters in the beam path fromthe flow cell 425 to each detector. Different fluorescent molecules usedfor a flow cytometer experiment will emit light in their owncharacteristic wavelength bands. The particular fluorescent labels usedfor an experiment and their associated fluorescent emission bands may beselected to generally coincide with the filter windows of the detectors.However, as more detectors are provided, and more labels are utilized,perfect correspondence between filter windows and fluorescent emissionspectra is not possible. It is generally true that although the peak ofthe emission spectra of a particular fluorescent molecule may lie withinthe filter window of one particular detector, some of the emissionspectra of that label will also overlap the filter windows of one ormore other detectors. This may be referred to as spillover. The I/O 497can be configured to receive data regarding a flow cytometer experimenthaving a panel of fluorescent labels and a plurality of cell populationshaving a plurality of markers, each cell population having a subset ofthe plurality of markers. The I/O 497 can also be configured to receivebiological data assigning one or more markers to one or more cellpopulations, marker density data, emission spectrum data, data assigninglabels to one or more markers, and cytometer configuration data. Flowcytometer experiment data, such as label spectral characteristics andflow cytometer configuration data can also be stored in the memory 495.The controller/processor 490 can be configured to evaluate one or moreassignments of labels to markers.

FIG. 5 shows a functional block diagram for one example of a particleanalyzer control system, such as an analytics controller 500, foranalyzing and displaying biological events. An analytics controller 500can be configured to implement a variety of processes for controllinggraphic display of biological events.

A particle analyzer or sorting system 502 can be configured to acquirebiological event data. For example, a flow cytometer can generate flowcytometric event data. The particle analyzer 502 can be configured toprovide biological event data to the analytics controller 500. A datacommunication channel can be included between the particle analyzer orsorting system 502 and the analytics controller 500. The biologicalevent data can be provided to the analytics controller 500 via the datacommunication channel.

The analytics controller 500 can be configured to receive biologicalevent data from the particle analyzer or sorting system 502. Thebiological event data received from the particle analyzer or sortingsystem 502 can include flow cytometric event data. The analyticscontroller 500 can be configured to provide a graphical displayincluding a first plot of biological event data to a display device 506.The analytics controller 500 can be further configured to render aregion of interest as a gate around a population of biological eventdata shown by the display device 506, overlaid upon the first plot, forexample. In some embodiments, the gate can be a logical combination ofone or more graphical regions of interest drawn upon a single parameterhistogram or bivariate plot. In some embodiments, the display can beused to display particle parameters or saturated detector data.

The analytics controller 500 can be further configured to display thebiological event data on the display device 506 within the gatedifferently from other events in the biological event data outside ofthe gate. For example, the analytics controller 500 can be configured torender the color of biological event data contained within the gate tobe distinct from the color of biological event data outside of the gate.The display device 506 can be implemented as a monitor, a tabletcomputer, a smartphone, or other electronic device configured to presentgraphical interfaces.

The analytics controller 500 can be configured to receive a gateselection signal identifying the gate from a first input device. Forexample, the first input device can be implemented as a mouse 510. Themouse 510 can initiate a gate selection signal to the analyticscontroller 500 identifying the gate to be displayed on or manipulatedvia the display device 506 (e.g., by clicking on or in the desired gatewhen the cursor is positioned there). In some implementations, the firstdevice can be implemented as the keyboard 508 or other means forproviding an input signal to the analytics controller 500 such as atouchscreen, a stylus, an optical detector, or a voice recognitionsystem. Some input devices can include multiple inputting functions. Insuch implementations, the inputting functions can each be considered aninput device. For example, as shown in FIG. 5, the mouse 510 can includea right mouse button and a left mouse button, each of which can generatea triggering event.

The triggering event can cause the analytics controller 500 to alter themanner in which the data is displayed, which portions of the data isactually displayed on the display device 506, and/or provide input tofurther processing such as selection of a population of interest forparticle sorting.

In some embodiments, the analytics controller 500 can be configured todetect when gate selection is initiated by the mouse 510. The analyticscontroller 500 can be further configured to automatically modify plotvisualization to facilitate the gating process. The modification can bebased on the specific distribution of biological event data received bythe analytics controller 500.

The analytics controller 500 can be connected to a storage device 504.The storage device 504 can be configured to receive and store biologicalevent data from the analytics controller 500. The storage device 504 canalso be configured to receive and store flow cytometric event data fromthe analytics controller 500. The storage device 504 can be furtherconfigured to allow retrieval of biological event data, such as flowcytometric event data, by the analytics controller 500.

A display device 506 can be configured to receive display data from theanalytics controller 500. The display data can comprise plots ofbiological event data and gates outlining sections of the plots. Thedisplay device 506 can be further configured to alter the informationpresented according to input received from the analytics controller 500in conjunction with input from the particle analyzer 502, the storagedevice 504, the keyboard 508, and/or the mouse 510.

In some implementations, the analytics controller 500 can generate auser interface to receive example events for sorting. For example, theuser interface can include a control for receiving example events orexample images. The example events or images or an example gate can beprovided prior to collection of event data for a sample, or based on aninitial set of events for a portion of the sample.

In some embodiments, systems of interest include a particle sortersystem. FIG. 6A is a schematic drawing of a particle sorter system 600(e.g., the particle analyzer or sorting system 502) in accordance withone embodiment presented herein. In some embodiments, the particlesorter system 600 is a cell sorter system. As shown in FIG. 6A, a dropformation transducer 602 (e.g., piezo-oscillator) is coupled to a fluidconduit 601, which can be coupled to, can include, or can be, a nozzle603. Within the fluid conduit 601, sheath fluid 604 hydrodynamicallyfocuses a sample fluid 606 comprising particles 609 into a moving fluidcolumn 608 (e.g. a stream). Within the moving fluid column 608,particles 609 (e.g., cells) are lined up in single file to cross amonitored area 611 (e.g., where laser-stream intersect), irradiated byan irradiation source 612 (e.g., a laser). Vibration of the dropformation transducer 602 causes moving fluid column 608 to break into aplurality of drops 610, some of which contain particles 609.

In operation, a detection station 614 (e.g., an event detector)identifies when a particle of interest (or cell of interest) crosses themonitored area 611. Detection station 614 feeds into a timing circuit628, which in turn feeds into a flash charge circuit 630. At a dropbreak off point, informed by a timed drop delay (Δt), a flash charge canbe applied to the moving fluid column 608 such that a drop of interestcarries a charge. The drop of interest can include one or more particlesor cells to be sorted. The charged drop can then be sorted by activatingdeflection plates (not shown) to deflect the drop into a vessel such asa collection tube or a multi-well or microwell sample plate where a wellor microwell can be associated with drops of particular interest. Asshown in FIG. 6A, the drops can be collected in a drain receptacle 638.

A detection system 616 (e.g. a drop boundary detector) serves toautomatically determine the phase of a drop drive signal when a particleof interest passes the monitored area 611. An exemplary drop boundarydetector is described in U.S. Pat. No. 7,679,039, which is incorporatedherein by reference in its entirety. The detection system 616 allows theinstrument to accurately calculate the place of each detected particlein a drop. The detection system 616 can feed into an amplitude signal620 and/or phase 618 signal, which in turn feeds (via amplifier 622)into an amplitude control circuit 626 and/or frequency control circuit624. The amplitude control circuit 626 and/or frequency control circuit624, in turn, controls the drop formation transducer 602. The amplitudecontrol circuit 626 and/or frequency control circuit 624 can be includedin a control system.

In some implementations, sort electronics (e.g., the detection system616, the detection station 614 and a processor 640) can be coupled witha memory configured to store the detected events and a sort decisionbased thereon. The sort decision can be included in the event data for aparticle. In some implementations, the detection system 616 and thedetection station 614 can be implemented as a single detection unit orcommunicatively coupled such that an event measurement can be collectedby one of the detection system 616 or the detection station 614 andprovided to the non-collecting element.

FIG. 6B is a schematic drawing of a particle sorter system, inaccordance with one embodiment presented herein. The particle sortersystem 600 shown in FIG. 6B, includes deflection plates 652 and 654. Acharge can be applied via a stream-charging wire in a barb. This createsa stream of droplets 610 containing particles 610 for analysis. Theparticles can be illuminated with one or more light sources (e.g.,lasers) to generate light scatter and fluorescence information. Theinformation for a particle is analyzed such as by sorting electronics orother detection system (not shown in FIG. 6B). The deflection plates 652and 654 can be independently controlled to attract or repel the chargeddroplet to guide the droplet toward a destination collection receptacle(e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, thedeflection plates 652 and 654 can be controlled to direct a particlealong a first path 662 toward the receptacle 674 or along a second path668 toward the receptacle 678. If the particle is not of interest (e.g.,does not exhibit scatter or illumination information within a specifiedsort range), deflection plates may allow the particle to continue alonga flow path 664. Such uncharged droplets may pass into a wastereceptacle such as via aspirator 670.

The sorting electronics can be included to initiate collection ofmeasurements, receive fluorescence signals for particles, and determinehow to adjust the deflection plates to cause sorting of the particles.Example implementations of the embodiment shown in FIG. 6B include theBD FACSAria™ line of flow cytometers commercially provided by Becton,Dickinson and Company (Franklin Lakes, N.J.).

Computer-Controlled Systems

Aspects of the present disclosure further include computer-controlledsystems, where the systems further include one or more computers forcomplete automation or partial automation. In some embodiments, systemsinclude a computer having a computer readable storage medium with acomputer program stored thereon, where the computer program when loadedon the computer includes instructions for irradiating a photodetectorwith a light source at a first intensity for a first predetermined timeinterval, irradiating the photodetector with the light source at asecond intensity for a second predetermined time interval, integratingdata signals from the photodetector over a period of time that includesthe first predetermined interval and the second predetermined interval;and determining one or more parameters of the photodetector based on theintegrated data signals. In some embodiments, systems include a computerhaving a computer readable storage medium with a computer program storedthereon, where the computer program when loaded on the computer includesinstructions for increasing the intensity of light from the light sourceover the period of time that includes at least the first predeterminedinterval and the second predetermined interval. In some instances, thecomputer program includes instructions for linearly increasing intensityof light from the light source. In some instances, the computer programincludes instructions for exponentially increasing intensity of lightfrom the light source.

In some embodiments, the computer program includes instructions tocalculate signal amplitude from the integrated data signals and forcomparing the calculated signal amplitude with the intensity ofirradiation by the light source. In certain embodiments, the computerprogram includes instructions to determine one or more of minimumdetection threshold, maximal detection threshold, detector sensitivity,detector dynamic range, detector signal-to-noise ratio and number ofphotoelectrons per unit output of the irradiated photodetector. Incertain instances, the computer program includes instructions todetermine the parameters of the photodetector over an operating voltagerange of the photodetector, such as over the entire operating voltagerange of the photodetector.

In embodiments, the system includes an input module, a processing moduleand an output module. The subject systems may include both hardware andsoftware components, where the hardware components may take the form ofone or more platforms, e.g., in the form of servers, such that thefunctional elements, i.e., those elements of the system that carry outspecific tasks (such as managing input and output of information,processing information, etc.) of the system may be carried out by theexecution of software applications on and across the one or morecomputer platforms represented of the system.

Systems may include a display and operator input device. Operator inputdevices may, for example, be a keyboard, mouse, or the like. Theprocessing module includes a processor which has access to a memoryhaving instructions stored thereon for performing the steps of thesubject methods. The processing module may include an operating system,a graphical user interface (GUI) controller, a system memory, memorystorage devices, and input-output controllers, cache memory, a databackup unit, and many other devices. The processor may be a commerciallyavailable processor or it may be one of other processors that are orwill become available. The processor executes the operating system andthe operating system interfaces with firmware and hardware in awell-known manner, and facilitates the processor in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages, such as Java, Perl, C++, otherhigh level or low level languages, as well as combinations thereof, asis known in the art. The operating system, typically in cooperation withthe processor, coordinates and executes functions of the othercomponents of the computer. The operating system also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques. The processor may be any suitableanalog or digital system. In some embodiments, processors include analogelectronics which allows the user to manually align a light source withthe flow stream based on the first and second light signals. In someembodiments, the processor includes analog electronics which providefeedback control, such as for example negative feedback control.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, flash memorydevices, or other memory storage device. The memory storage device maybe any of a variety of known or future devices, including a compact diskdrive, a tape drive, a removable hard disk drive, or a diskette drive.Such types of memory storage devices typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, or floppy diskette.Any of these program storage media, or others now in use or that maylater be developed, may be considered a computer program product. Aswill be appreciated, these program storage media typically store acomputer software program and/or data. Computer software programs, alsocalled computer control logic, typically are stored in system memoryand/or the program storage device used in conjunction with the memorystorage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor the computer, causes the processor to performfunctions described herein. In other embodiments, some functions areimplemented primarily in hardware using, for example, a hardware statemachine. Implementation of the hardware state machine so as to performthe functions described herein will be apparent to those skilled in therelevant arts.

Memory may be any suitable device in which the processor can store andretrieve data, such as magnetic, optical, or solid-state storage devices(including magnetic or optical disks or tape or RAM, or any othersuitable device, either fixed or portable). The processor may include ageneral-purpose digital microprocessor suitably programmed from acomputer readable medium carrying necessary program code. Programmingcan be provided remotely to processor through a communication channel,or previously saved in a computer program product such as memory or someother portable or fixed computer readable storage medium using any ofthose devices in connection with memory. For example, a magnetic oroptical disk may carry the programming, and can be read by a diskwriter/reader. Systems of the invention also include programming, e.g.,in the form of computer program products, algorithms for use inpracticing the methods as described above. Programming according to thepresent invention can be recorded on computer readable media, e.g., anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM;portable flash drive; and hybrids of these categories such asmagnetic/optical storage media.

The processor may also have access to a communication channel tocommunicate with a user at a remote location. By remote location ismeant the user is not directly in contact with the system and relaysinput information to an input manager from an external device, such as acomputer connected to a Wide Area Network (“WAN”), telephone network,satellite network, or any other suitable communication channel,including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may beconfigured to include a communication interface. In some embodiments,the communication interface includes a receiver and/or transmitter forcommunicating with a network and/or another device. The communicationinterface can be configured for wired or wireless communication,including, but not limited to, radio frequency (RF) communication (e.g.,Radio-Frequency Identification (RFID), Zigbee communication protocols,WiFi, infrared, wireless Universal Serial Bus (USB), Ultra Wide Band(UWB), Bluetooth® communication protocols, and cellular communication,such as code division multiple access (CDMA) or Global System for Mobilecommunications (GSM).

In one embodiment, the communication interface is configured to includeone or more communication ports, e.g., physical ports or interfaces suchas a USB port, an RS-232 port, or any other suitable electricalconnection port to allow data communication between the subject systemsand other external devices such as a computer terminal (for example, ata physician's office or in hospital environment) that is configured forsimilar complementary data communication.

In one embodiment, the communication interface is configured forinfrared communication, Bluetooth® communication, or any other suitablewireless communication protocol to enable the subject systems tocommunicate with other devices such as computer terminals and/ornetworks, communication enabled mobile telephones, personal digitalassistants, or any other communication devices which the user may use inconjunction.

In one embodiment, the communication interface is configured to providea connection for data transfer utilizing Internet Protocol (IP) througha cell phone network, Short Message Service (SMS), wireless connectionto a personal computer (PC) on a Local Area Network (LAN) which isconnected to the internet, or WiFi connection to the internet at a WiFihotspot.

In one embodiment, the subject systems are configured to wirelesslycommunicate with a server device via the communication interface, e.g.,using a common standard such as 802.11 or Bluetooth® RF protocol, or anIrDA infrared protocol. The server device may be another portabledevice, such as a smart phone, Personal Digital Assistant (PDA) ornotebook computer; or a larger device such as a desktop computer,appliance, etc. In some embodiments, the server device has a display,such as a liquid crystal display (LCD), as well as an input device, suchas buttons, a keyboard, mouse or touch-screen.

In some embodiments, the communication interface is configured toautomatically or semi-automatically communicate data stored in thesubject systems, e.g., in an optional data storage unit, with a networkor server device using one or more of the communication protocols and/ormechanisms described above.

Output controllers may include controllers for any of a variety of knowndisplay devices for presenting information to a user, whether a human ora machine, whether local or remote. If one of the display devicesprovides visual information, this information typically may be logicallyand/or physically organized as an array of picture elements. A graphicaluser interface (GUI) controller may include any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between the system and a user, and for processing userinputs. The functional elements of the computer may communicate witheach other via system bus. Some of these communications may beaccomplished in alternative embodiments using network or other types ofremote communications. The output manager may also provide informationgenerated by the processing module to a user at a remote location, e.g.,over the Internet, phone or satellite network, in accordance with knowntechniques. The presentation of data by the output manager may beimplemented in accordance with a variety of known techniques. As someexamples, data may include SQL, HTML or XML documents, email or otherfiles, or data in other forms. The data may include Internet URLaddresses so that a user may retrieve additional SQL, HTML, XML, orother documents or data from remote sources. The one or more platformspresent in the subject systems may be any type of known computerplatform or a type to be developed in the future, although theytypically will be of a class of computer commonly referred to asservers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known orfuture type of cabling or other communication system including wirelesssystems, either networked or otherwise. They may be co-located or theymay be physically separated. Various operating systems may be employedon any of the computer platforms, possibly depending on the type and/ormake of computer platform chosen. Appropriate operating systems includeWindows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux,OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others.

FIG. 7 depicts a general architecture of an example computing device 700according to certain embodiments. The general architecture of thecomputing device 700 depicted in FIG. 7 includes an arrangement ofcomputer hardware and software components. The computing device 700 mayinclude many more (or fewer) elements than those shown in FIG. 7. It isnot necessary, however, that all of these generally conventionalelements be shown in order to provide an enabling disclosure. Asillustrated, the computing device 700 includes a processing unit 710, anetwork interface 720, a computer readable medium drive 730, aninput/output device interface 740, a display 750, and an input device760, all of which may communicate with one another by way of acommunication bus. The network interface 720 may provide connectivity toone or more networks or computing systems. The processing unit 710 maythus receive information and instructions from other computing systemsor services via a network. The processing unit 710 may also communicateto and from memory 770 and further provide output information for anoptional display 750 via the input/output device interface 740. Theinput/output device interface 740 may also accept input from theoptional input device 760, such as a keyboard, mouse, digital pen,microphone, touch screen, gesture recognition system, voice recognitionsystem, gamepad, accelerometer, gyroscope, or other input device.

The memory 770 may contain computer program instructions (grouped asmodules or components in some embodiments) that the processing unit 710executes in order to implement one or more embodiments. The memory 770generally includes RAM, ROM and/or other persistent, auxiliary ornon-transitory computer-readable media. The memory 770 may store anoperating system 772 that provides computer program instructions for useby the processing unit 710 in the general administration and operationof the computing device 700. The memory 770 may further include computerprogram instructions and other information for implementing aspects ofthe present disclosure.

Computer-Readable Storage Medium

Aspects of the present disclosure further include non-transitorycomputer readable storage mediums having instructions for practicing thesubject methods. Computer readable storage mediums may be employed onone or more computers for complete automation or partial automation of asystem for practicing methods described herein. In certain embodiments,instructions in accordance with the method described herein can be codedonto a computer-readable medium in the form of “programming”, where theterm “computer readable medium” as used herein refers to anynon-transitory storage medium that participates in providinginstructions and data to a computer for execution and processing.Examples of suitable non-transitory storage media include a floppy disk,hard disk, optical disk, magneto-optical disk, CD-ROM, CD-R, magnetictape, non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid statedisk, and network attached storage (NAS), whether or not such devicesare internal or external to the computer. A file containing informationcan be “stored” on computer readable medium, where “storing” meansrecording information such that it is accessible and retrievable at alater date by a computer. The computer-implemented method describedherein can be executed using programming that can be written in one ormore of any number of computer programming languages. Such languagesinclude, for example, Java (Sun Microsystems, Inc., Santa Clara,Calif.), Visual Basic (Microsoft Corp., Redmond, Wash.), and C++ (AT&TCorp., Bedminster, N.J.), as well as any many others.

In some embodiments, computer readable storage media of interest includea computer program stored thereon, where the computer program whenloaded on the computer includes instructions having: algorithm forirradiating a photodetector with a continuous wave light source at afirst intensity for a first predetermined time interval; algorithm forirradiating the photodetector with the light source at a secondintensity for a second predetermined time interval; algorithm forintegrating data signals from the photodetector over a period of timecomprising the first predetermined interval and the second predeterminedinterval; and algorithm for determining one or more parameters of thephotodetector based on the integrated data signals.

In certain instances, the non-transitory computer readable storagemedium includes algorithm for irradiating the photodetector with aplurality of light intensities over a plurality of time intervals. Inthese instances, the non-transitory computer readable storage mediumincludes algorithm for integrating data signals from the photodetectorover a time period that includes the plurality of irradiation timeintervals.

In some embodiments, the non-transitory computer readable storage mediumincludes algorithm for calculating a signal amplitude. In someinstances, the non-transitory computer readable storage medium includesalgorithm for calculating the median signal amplitude. In certaininstances, the non-transitory computer readable storage medium includesalgorithm for comparing the calculated signal amplitude with the lightintensity of the light source. In certain instance, the non-transitorycomputer readable storage medium includes algorithm for determining aparameter of the photodetector based on one or more of the calculatedsignal amplitude and the comparison between the calculated signalamplitude with the light intensity of the light source. For example, thenon-transitory computer readable storage medium may include algorithmfor determining minimum detection threshold, maximal detectionthreshold, detector sensitivity, detector dynamic range, detectorsignal-to-noise ratio or number of photoelectrons per unit output. Thenon-transitory computer readable storage medium may include algorithmfor determining the detector parameter over a range of operatingvoltages of the photodetector, such as where the parameters of thephotodetector are determined over the entire operating voltage range ofthe photodetector.

The computer readable storage medium may be employed on one or morecomputer systems having a display and operator input device. Operatorinput devices may, for example, be a keyboard, mouse, or the like. Theprocessing module includes a processor which has access to a memoryhaving instructions stored thereon for performing the steps of thesubject methods. The processing module may include an operating system,a graphical user interface (GUI) controller, a system memory, memorystorage devices, and input-output controllers, cache memory, a databackup unit, and many other devices. The processor may be a commerciallyavailable processor or it may be one of other processors that are orwill become available. The processor executes the operating system andthe operating system interfaces with firmware and hardware in awell-known manner, and facilitates the processor in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages, such as Java, Perl, C++, otherhigh level or low level languages, as well as combinations thereof, asis known in the art. The operating system, typically in cooperation withthe processor, coordinates and executes functions of the othercomponents of the computer. The operating system also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques.

Multi-Spectral Fluorescent Particles

As summarized above, aspects of the present disclosure also includeparticles (e.g., beads) having one or more fluorophores for practicingcertain methods described herein. Particles of interest according tocertain embodiments may include a single-peak multi-fluorophore beadthat provides for a bright photodetector signal across all light sourcewavelengths (e.g., across all LEDs or lasers of the system) and acrossdetection wavelengths of the photodetectors.

In embodiments, the subject particles are formulated (e.g., in a fluidiccomposition) for flowing in a flow stream irradiated by a light sourceas described above. Each particle may have one or more different typesof fluorophores, such as 2 or more, or 3 or more, or 4 or more, or 5 ormore, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 ormore, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15or more, 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20or more, or 25 or more, or 30 or more, or 35 or more, or 40 or more, or45 or more, 50 or more different types of fluorophores. For example,each particle may include 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9,or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or20 different types of fluorophores.

In embodiments, each fluorophore is stably associated with the particle.By stably associated is meant that the fluorophore does not readilydissociate from the particle to contact with a liquid medium, e.g., anaqueous medium. In some embodiments, one or more of the fluorophores arecovalently conjugated to the particle. In other embodiments, one or moreof the fluorophores are physically associated (i.e., non-covalentlycoupled) to the particle. In other embodiments, one or more fluorophoresare covalently conjugated to the particle and one or more fluorophoresare physically associated with the particle.

In some embodiments, each particle includes 2 or more different types offluorophores. Any two fluorophores are considered to be different andistinct if they differ from each other by one or more of molecularformula, excitation maximum and emission maximum. As such, different ordistinct fluorophores may differ from each other in terms of chemicalcomposition or in terms of one or more properties of the fluorophore.For instance, different fluorophores may differ from each other by atleast one of excitation maxima and emission maxima. In some cases,different fluorophores differ from each other by their excitationmaxima. In some cases, different fluorophores differ from each other bytheir emission maxima. In some cases, different fluorophores differ fromeach other by both their excitation maxima and emission maxima. As such,in embodiments that include first and second fluorophores, the first andsecond fluorophore may differ from each other by at least one ofexcitation maxima and emission maxima. For example, the first and secondfluorophore may differ from each other by excitation maxima, by emissionmaxima, or by both excitation and emission maxima. A given set offluorophores may be considered distinct if they differ from each otherin terms of excitation or emission maximum, where the magnitude of suchdifference is, in some instances, 5 nm or more, such 10 nm or more,including 15 nm or more, wherein in some instances the magnitude of thedifference ranges from 5 to 400 nm, such as 10 to 200 nm, including 15to 100 nm, such as 25 to 50 nm.

Fluorophores of interest according to certain embodiments haveexcitation maxima that range from 100 nm to 800 nm, such as from 150 nmto 750 nm, such as from 200 nm to 700 nm, such as from 250 nm to 650 nm,such as from 300 nm to 600 nm and including from 400 nm to 500 nm.Fluorophores of interest according to certain embodiments have emissionmaxima that range from 400 nm to 1000 nm, such as from 450 nm to 950 nm,such as from 500 nm to 900 nm, such as from 550 nm to 850 nm andincluding from 600 nm to 800 nm. In certain instances, the fluorophoreis a light emitting dye such as a fluorescent dye having a peak emissionwavelength of 200 nm or more, such as 250 nm or more, such as 300 nm ormore, such as 350 nm or more, such as 400 nm or more, such as 450 nm ormore, such as 500 nm or more, such as 550 nm or more, such as 600 nm ormore, such as 650 nm or more, such as 700 nm or more, such as 750 nm ormore, such as 800 nm or more, such as 850 nm or more, such as 900 nm ormore, such as 950 nm or more, such as 1000 nm or more and including 1050nm or more. For example, the fluorophore may be a fluorescent dye havinga peak emission wavelength that ranges from 200 nm to 1200 nm, such asfrom 300 nm to 1100 nm, such as from 400 nm to 1000 nm, such as from 500nm to 900 nm and including a fluorescent dye having a peak emissionwavelength of from 600 nm to 800 nm. In certain embodiments, the subjectmultispectral particles provide for stable excitation by lasers whichirradiate at a wavelength at or about 349 nm (UV laser), 488 nm (bluelaser), 532 nm (Nd:YAG solid state laser), 640 nm (red laser) and 405 nm(violet laser). In certain instances, the subject multispectralparticles provide for stable excitation by a light source across a fullspectral detection band, such as from 350 nm to 850 nm.

Fluorophores of interest may include, but are not limited to, a bodipydye, a coumarin dye, a rhodamine dye, an acridine dye, an anthraquinonedye, an arylmethane dye, a diarylmethane dye, a chlorophyll containingdye, a triarylmethane dye, an azo dye, a diazonium dye, a nitro dye, anitroso dye, a phthalocyanine dye, a cyanine dye, an asymmetric cyaninedye, a quinon-imine dye, an azine dye, an eurhodin dye, a safranin dye,an indamin, an indophenol dye, a fluorine dye, an oxazine dye, anoxazone dye, a thiazine dye, a thiazole dye, a xanthene dye, a fluorenedye, a pyronin dye, a fluorine dye, a rhodamine dye, a phenanthridinedye, squaraines, bodipys, squarine roxitanes, naphthalenes, coumarins,oxadiazoles, anthracenes, pyrenes, acridines, arylmethines, ortetrapyrroles and a combination thereof. In certain embodiments,conjugates may include two or more dyes, such as two or more dyesselected from a bodipy dye, a coumarin dye, a rhodamine dye, an acridinedye, an anthraquinone dye, an arylmethane dye, a diarylmethane dye, achlorophyll containing dye, a triarylmethane dye, an azo dye, adiazonium dye, a nitro dye, a nitroso dye, a phthalocyanine dye, acyanine dye, an asymmetric cyanine dye, a quinon-imine dye, an azinedye, an eurhodin dye, a safranin dye, an indamin, an indophenol dye, afluorine dye, an oxazine dye, an oxazone dye, a thiazine dye, a thiazoledye, a xanthene dye, a fluorene dye, a pyronin dye, a fluorine dye, arhodamine dye, a phenanthridine dye, squaraines, bodipys, squarineroxitanes, naphthalenes, coumarins, oxadiazoles, anthracenes, pyrenes,acridines, arylmethines, or tetrapyrroles and a combination thereof.

In certain embodiments, fluorophores of interest may include but are notlimited to fluorescein isothiocyanate (FITC), a phycoerythrin (PE) dye,a peridinin chlorophyll protein-cyanine dye (e.g., PerCP-Cy5.5), aphycoerythrin-cyanine (PE-Cy) dye (PE-Cy7), an allophycocyanin (APC) dye(e.g., APC-R700), an allophycocyanin-cyanine dye (e.g., APC-Cy7), acoumarin dye (e.g., V450 or V500). In certain instances, fluorophoresmay include one or more of 1,4-bis-(o-methylstyryl)-benzene (bis-MSB1,4-bis[2-(2-methylphenyl)ethenyl]-benzene), a C510 dye, a C6 dye, nilered dye, a T614 dye (e.g.,N-[7-(methanesulfonamido)-4-oxo-6-phenoxychromen-3-yl]formamide), LDS821 dye((2-(6-(p-dimethylaminophenyl)-2,4-neopentylene-1,3,5-hexatrienyl)-3-ethylbenzothiazoliumperchlorate), an mFluor dye (e.g., an mFluor Red dye such as mFluor780NS).

The particles may be any convenient shape for irradiating by the lightsource as described above. In some instances the particle is a solidsupport that is shaped or configured as discs, spheres, ovates, cubes,blocks, cones, etc., as well as irregular shapes. The mass of theparticles may vary, ranging in some instances from 0.01 mg to 20 mg,such as from 0.05 mg to 19.5 mg, such as from 0.1 mg to 19 mg, such asfrom 0.5 mg to 18.5 mg, such as from 1 mg to 18 mg, such as from 1.5 mgto 17.5 mg, such as from 2 mg to 15 mg and including from 3 mg to 10 mg.The particle may have a surface area of 0.01 mm² or more, such as 0.05mm² or more, such as 0.1 mm² or more, such as 0.5 mm² or more, such as 1mm² or more, such as 1.5 mm² or more, such as 2 mm² or more, such as 2.5mm² or more, such as 3 mm² or more, such as 3.5 mm² or more, such as 4mm² or more, such as 4.5 mm² or more and including 5 mm² or more, e.g.,as determined using a Vertex system or equivalent.

The dimensions of the particles may vary, as desired, where in someinstances, particles have a longest dimension ranging from 0.01 mm to 10mm, such as from 0.05 mm to 9.5 mm, such as from 0.1 mm to 9 mm, such asfrom 0.5 mm to 8.5 mm, such as from 1 mm to 8 mm, such as from 1.5 mm to7.5 mm, such as from 2 mm to 7 mm, such as from 2.5 mm to 6.5 mm andincluding from 3 mm to 6 mm. In certain instances, particles have ashortest dimension ranging from 0.01 mm to 5 mm, such as from 0.05 mm to4.5 mm, such as from 0.1 mm to 4 mm, such as from 0.5 mm to 3.5 mm andincluding from 1 mm to 3 mm.

In certain instances, particles of interest are porous, such as wherethe particles have a porosity ranging from 5μ to 100μ, such as from 10μto 90μ, such as from 15μ to 85μ, such as from 20μ to 80μ, such as from25μ to 75μ and including from 30μ to 70μ, for instance 50μ as determinedfor example using a Capillary Flow Porometer or equivalent.

The particles may be formed from any convenient material. Of interest insome embodiments are particles, e.g., beads, having low or noauto-fluorescence. Suitable materials include, but are not limited to,glass materials (e.g., silicates), ceramic materials (e.g., calciumphosphates), metallic materials, and polymeric materials, etc. such asfor example, polyethylene, polypropylene, polytetrafluoroethylene,polyvinylidine fluoride, and the like. In some instances, the particlesare formed from a solid support, such as the porous matrices asdescribed in U.S. Published Application Publication No. U.S. Pat. No.9,797,899, the disclosure of which is herein incorporated by reference.As such, a surface area of the particle may be any suitable macroporousor microporous substrate, where suitable macroporous and microporoussubstrates include, but are not limited to, ceramic matrices, frits,such as fritted glass, polymeric matrices as well as metal-organicpolymeric matrices. In some embodiments, the porous matrix is a frit.The term “frit” is used herein in its conventional sense to refer to theporous composition formed from a sintered granulated solid, such asglass. Frits may have a chemical constituent which vary, depending onthe type of sintered granulate used to prepare the frit, where fritsthat may be employed include, but are not limited to, frits composed ofaluminosilicate, boron trioxide, borophosphosilicate glass, borosilicateglass, ceramic glaze, cobalt glass, cranberry glass, fluorophosphateglass, fluorosilicate glass, fuzed quartz, germanium dioxide, metal andsulfide embedded borosilicate, leaded glass, phosphate glass, phosphoruspentoxide glass, phosphosilicate glass, potassium silicate, soda-limeglass, sodium hexametaphosphate glass, sodium silicate, tellurite glass,uranium glass, vitrite and combinations thereof. In some embodiments,the porous matrix is a glass frit, such as a borosilicate,aluminosilicate, fluorosilicate, potassium silicate orborophosphosilicate glass frit.

In some embodiments, the particle is formed from a porous organicpolymer. Porous organic polymers of interest vary depending on thesample volume, components in the sample as well as assay reagent presentand may include but are not limited to porous polyethylene,polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride(PVDF), ethyl vinyl acetate (EVA), polycarbonate, polycarbonate alloys,polyurethane, polyethersulfone, copolymers and combinations thereof. Forexample, porous polymers of interest include homopolymers,heteropolymers and copolymers composed of monomeric units such asstyrene, monoalkylene allylene monomers such as ethyl styrene, α-methylstyrene, vinyl toluene, and vinyl ethyl benzene; (meth)acrylic esterssuch as methyl(meth)acrylate, ethyl(meth)acrylate, butyl(meth)acrylate,isobutyl(meth)acrylate, isodecyl(meth)acrylate, 2-ethylhexyl(meth)acrylate, lauryl(meth)acrylate, stearyl(meth)acrylate,cyclohexyl(meth)acrylate, and benzyl(meth)acrylate; chlorine-containingmonomers such as vinyl chloride, vinylidenechloride, andchloromethylstyrene; acrylonitrile compounds such as acrylonitrile andmethacrylonitrile; and vinyl acetate, vinyl propionate, n-octadecylacrylamide, ethylene, propylene, and butane, and combinations thereof.

In some embodiments, the particles are formed from a metal organicpolymer matrix, for example an organic polymer matrix that has abackbone structure that contains a metal such as aluminum, barium,antimony, calcium, chromium, copper, erbium, germanium, iron, lead,lithium, phosphorus, potassium, silicon, tantalum, tin, titanium,vanadium, zinc or zirconium. In some embodiments, the porous metalorganic matrix is an organosiloxane polymer including but not limited topolymers of methyltrimethoxysilane, dimethyldimethoxysilane,tetraethoxysilane, methacryloxypropyltrimethoxysilane,bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane,bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane,bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinationsthereof.

Kits

Kits including one or more components of the subject systems are alsoprovided. Kits according to certain embodiments include one or morecontinuous wave light source, such as a narrow band light emitting diodeand a photodetector (e.g., a photomultiplier tube) where analysis of oneor more parameters of the photodetector is desired. Kits may alsoinclude an optical adjustment component, such as lenses, mirrors,filters, fiber optics, wavelength separators, pinholes, slits,collimating protocols and combinations thereof.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), portable flash drive, and the like, on which the information hasbeen recorded. Yet another form of these instructions that may bepresent is a website address which may be used via the internet toaccess the information at a removed site.

Utility

The subject methods, systems and computer systems find use in a varietyof applications where it is desirable to calibrate or optimize aphotodetector, such as in a particle analyzer. The subject methods andsystems also find use for photodetectors that are used to analyze andsort particle components in a sample in a fluid medium, such as abiological sample. The present disclosure also finds use in flowcytometry where it is desirable to provide a flow cytometer withimproved cell sorting accuracy, enhanced particle collection, reducedenergy consumption, particle charging efficiency, more accurate particlecharging and enhanced particle deflection during cell sorting. Inembodiments, the present disclosure reduces the need for user input ormanual adjustment during sample analysis with a flow cytometer. Incertain embodiments, the subject methods and systems provide fullyautomated protocols so that adjustments to a flow cytometer during userequire little, if any human input.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

1. A method for determining a parameter of a photodetector in a particleanalyzer, the method comprising: irradiating a photodetector positionedin a particle analyzer with a light source at a first intensity for afirst predetermined time interval; irradiating the photodetector withthe light source at a second intensity for a second predetermined timeinterval; integrating data signals from the photodetector over a periodof time comprising the first predetermined interval and the secondpredetermined interval; and determining one or more parameters of thephotodetector based on the integrated data signals.
 2. The methodaccording to claim 1, wherein the particle analyzer is incorporated in aflow cytometer.
 3. The method according to claim 1, wherein thephotodetector is positioned in the particle analyzer to detect lightfrom particles in a flow stream.
 4. The method according to claim 1,wherein the light source is a continuous wave light source.
 5. Themethod according to claim 1, wherein the light source is a pulsed lightsource.
 6. The method according to claim 1, wherein the light source isa light emitting diode.
 7. The method according to claim 1, wherein thelight source is a narrow bandwidth light source.
 8. The method accordingto claim 7, wherein the light source emits light comprising wavelengthsthat span 20 nm or less.
 9. The method according to claim 1, wherein themethod comprises irradiating the photodetector with a second intensitythat is greater than the first intensity.
 10. The method according toclaim 1, wherein the first predetermined time interval and the secondpredetermined time interval have the same duration.
 11. The methodaccording to claim 1, wherein the method comprises continuouslyirradiating the photodetector over the period of time comprising thefirst predetermined interval and the second predetermined interval. 12.The method according to claim 1, wherein the method comprises increasingthe intensity of light from the light source from the first intensity tothe second intensity over a third predetermined time interval.
 13. Themethod according to claim 1, wherein the method comprises increasing theintensity of light from the light source over the period of timecomprising the first predetermined interval and the second predeterminedinterval. 14-15. (canceled)
 16. The method according to claim 1, whereinintegrating data signals from the photodetector comprises calculatingsignal amplitude over the period of time.
 17. The method according toclaim 16, wherein the method comprises calculating a median signalamplitude over the period of time.
 18. (canceled)
 19. The methodaccording to claim 1, wherein the method comprises determining one ormore parameters of the photodetector selected from the group consistingof minimum detection threshold, maximal detection threshold, detectorsensitivity, detector dynamic range, detector signal-to-noise ratio andnumber of photoelectrons per unit output.
 20. The method according toclaim 1, wherein the method comprises: irradiating the photodetectorwith the light source at a plurality of intensities over a plurality ofpredetermined time intervals; integrating data signals from thephotodetector over a period of time comprising the plurality ofpredetermined time intervals; and determining one or more parameters ofthe photodetector based on the integrated data signals.
 21. The methodaccording to claim 1, wherein the parameters of the photodetector aredetermined over a range of operating voltages of the photodetector. 22.The method according to claim 21, wherein the parameters of thephotodetector are determined over the entire operating voltage range ofthe photodetector.
 23. The method according to claim 1, furthercomprising calculating an optimal detector gain for the photodetectorbased on the determined parameters. 24-115. (canceled)